Methods for treating central nervous system damage

ABSTRACT

Methods for the treatment of CNS damage are described, and include inducing in a subject in need of such treatment, a therapeutically effective amount of functional electronic stimulation (FES) sufficient to evoke patterned movement in the subject&#39;s muscles, the control of which has been affected by the CNS damage. The induction of FES-evoked patterned movement at least partially restores lost motor and sensory function, and stimulates regeneration of neural progenitor cells in the subject patient.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application from U.S. application.Ser. No. 10/654,832 filed on Sep. 4, 2003, now U.S Pat. No. 7,610,096,which claims priority from Provisional Application Ser. No. 60/408,214filed on Sep. 4, 2002, each of which is incorporated herein by referencein their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with Government support under NationalInstitutes of Health, National Institute of Neurological Disorders andStroke Grant NS40520. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

This invention relates in general to the field of medicine and totherapeutic methods for treating disruption of neural function resultingfrom damage to the central nervous system (CNS), and more particularlyto methods for restoring motor and sensory function in subjectssuffering from CNS damage.

Damage to the mammalian CNS can produce devastating physical impairment,and incur many added medical complications that in themselves can belife-threatening. CNS damage can result, for example, from spinal cordinjury (“SCI”), which typically involves a complete or partial severanceof a region of the spinal cord in an auto or other accident, or from astroke-induced lesion. SCI can also be the result of a chronic diseaseprocess such as multiple sclerosis or a cancerous tumor. Acute traumaand chronic disease processes such as multiple sclerosis can alsoproduce lesions elsewhere in the CNS that result in dramatic impairmentof sensory or motor function, or both.

Besides impairment of motor and sensory function, additional medicalconsequences of CNS damage almost always arise. For example, medicalcomplications resulting from substantial motor impairment includemuscular atrophy, loss of bone mineral content, decubitus ulcers,urinary tract infections, muscle spasticity, impaired circulation, andreduced heart and lung capacity.

The American Spinal Injury Association (“ASIA”) has developedinternational standards for examining and reporting the severity ofspinal cord injury (“SCI”). Table 1 presents ASIA's five grades ofspinal cord function, A to E, where E is normal. The ASIA Grade Adescribes individuals with the least remaining function. Such patientshave little hope for recovery. Less than 1% of those with no muscleactivity in the lower extremities 1 month after injury learn to walkagain, and only 10% recover enough function to be reclassified as ASIA Bor better. Approximately 90% of patients remain classified as ASIA GradeA.

TABLE 1 ASIA's international standards for classifying SCI GradeDescription A Complete. No sensory or motor function preserved in sacralsegments S4-S5 B Incomplete. Sensory but not motor function preservedbelow the neurologic level and extending through sacral segments S4-S5 CIncomplete. Motor function preserved below the neurologic level;majority of key muscle have a grade <3 D Incomplete. Motor functionpreserved below the neurologic level; majority of key muscles have agrade >3 E Normal motor and sensory function

Patients with SCI are often told that improvement or recovery occurslargely in the first 6 months after injury and is complete by 2 years.Indeed, the literature does not provide a single example of anindividual with an ASIA Grade A SCI who recovered by more than one grade2 years after injury. Some delayed recoveries occur, but the timeframeis typically between 1 and 6 months after injury for large improvements.Such recoveries are most common when an accompanying head injury impedesinitial progress. Small improvements can occur after periods longer than2 years but typically occur in individuals with incomplete injuries.

Some number of individuals with ASIA Grade A SCI might have at leastsome functional connections across the SCI lesion. Indeed, it is rarefor the cord to be completely severed by spinal trauma unless a gunshotor knife attack causes the wound. Studies performed in the 1950sindicate that limited preservation of white matter across the SCI lesioncan sustain substantial spinal cord function. Preservation of less than10% of the normal axon complement in the cat spinal cord can supportwalking, although this should not be viewed as the optimal requirement.Moreover, detailed anatomical postmortem studies of chronic SCI inhumans reveal that small residual connections across the lesion canpreserve some function. For example, one individual with ASIA Grade CSCI had retained only 1.17 mm² of white matter at the level of thelesion. Another patient with some preserved motor function below thelevel of a cervical injury had only 3175 corticospinal axons—less than8% of the number (41,472) found in normal controls.

A decade ago, the adult central nervous system (“CNS”) was thought to beincapable of regeneration. Rehabilitation focused primarily on theimmediate post-injury phase, when patients were still in the hospital,and aimed to maximize existing function and minimize complications.Although these goals are still important, the concept of spontaneousregeneration has emerged.

Current treatments for SCI are both limited and controversial.Administration of methylprednisolone within 8 hours of traumatic injurywas one of the first drug therapies to receive support. Limitations intechnical design and the marginal clinical effect of methylprednisoloneseen in the original multicenter studies have raised concern about thisapproach. Other medications, including Naloxone and Tirilazad, have alsobeen examined; however, those studied did not achieve their primaryendpoints. Most recently, the ganglioside, GM-1, has shown promise whenadministered in the subacute injury period, although primary endpointswere again not achieved. In general, most rehabilitation approaches areaccepted in the field but specific aspects have not been tested forefficacy in the SCI population. Moreover, the duration of inpatientrehabilitation has dramatically decreased over the last 10 years,necessitating the development of cost-effective in-home therapies.

Functional electrical Stimulation (“FES”) has been used to restorecertain functions following neural injury or disease. Examples includecochlear implants for restoration of hearing, stimulation of lower motorneurons to restore motor function including hand grasp and release,standing and stepping, breathing, and bladder emptying, and deep brainstimulation to treat the motor symptoms of Parkinson's disease (Grilland Kirsch, 1999; McDonald and Sadowsky, 2002). Each of theseapplications works by stimulating neural activity in existing neuronsand muscles to restore control to systems where control has beencompromised by injury or disease. However, FES has not been applied toreversing or altering disease progression or to promoting tissueregeneration or repair.

Locomotor training, such as on a treadmill, has been applied to motorincomplete patients (i.e., ASIA grade B or better) to enhance recoveryof walking. Locomotor training can help such patients improve theirwalking speed and posture, and to relearn and maintain balance whilewalking. These improvements correlate well with correspondingimprovements in muscle strength and in cardiovascular fitness, and arealso believed to involve improved recruitment of existing, spared motorneurons. Locomotor therapy has been combined with partial body weightsupport (BWS) of up to 40%, to reduce the load borne by lower limbsduring training, straighten walking posture, and assist certain aspectsof gait such as swing and balance. BWS of greater than about 30-40% isgenerally thought to substantially limit any benefit of locomotortraining to the patient.

FES therapy has been used in combination with locomotor or cyclingtraining for motor incomplete patients. Field-Fote describes thecombined use of FES with partial BWS and treadmill training, to improveover-ground walking ability in patients of ASIA grade C (Edelle C.Field-Fote, Arch. Phys. Med. Rehabil. 82: 818-24, (June 2001)). Patientswere able to support at least 70% of their own body weight and were ableto walk on a treadmill. Patients treated accordingly showed improvedover-ground walking speed in the absence of BWS and FES, due to trainingeffects on the musculoskeletal and cardiovascular systems. FES cyclinghas been described, for promoting recovery of leg strength and endurancein a motor incomplete patient (N. Donaldson et al., Spinal Cord38:680-82 (2000)). FES cycling in the single subject resulted inincreased leg muscle thickness and voluntary leg strength. However, onlymotor incomplete patients have been deemed to derive a benefit from FEStherapy, or from locomotor training, or from a combination of the two,because the benefits of such therapy are linked to the training effectson existing, spared (i.e. undamaged) elements of the patient'sneuromuscular system. Accordingly, since such training effects areobviated by the extreme injury and lack of sparing in motor completepatients, FES therapy and locomotor therapy have not even been triedwith such patients.

A great need therefore remains for novel approaches to partially orcompletely restoring lost or impaired sensory and motor function inindividuals suffering from disruptions of motor and sensory function dueto damage of the CNS.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel approach to restoring motor andsensory function in individuals suffering from CNS damage includingthose individuals with such extensive CNS damage that recovery of anyfunction requiring the activity of the lost or damaged neurons waspreviously thought impossible. The methods are based in part onsurprising findings in a motor complete human subject with a highcervical (C2) traumatic injury. The patient showed only spotty sensationin the left hemibody over the five years following the injury. Whentreated with a regimen of physical therapy that included application offunctional electrical stimulation (FES) sufficient to evoke patternedmovement in the subject's lower limbs, the subject showed unexpected,late partial recovery of motor function during years 6-8 post-injury.The methods are also based in part on complementary experimentalfindings in a rat model of spinal cord injury, which demonstrate thesurprising effects of the current methods on neural cell regenerationand on motor function in rat.

Accordingly, a method for treating central nervous system (CNS) damagein a mammalian subject in need of such treatment includes exposing themammalian subject to a therapeutically effective amount of functionalelectrical stimulation (FES)-evoked patterned movement. According to anexemplary embodiment, exposing the mammalian subject to atherapeutically effective amount of FES-evoked patterned movementincludes exposure for a period of time sufficient to regenerate neuralcells in the CNS of the subject.

In another embodiment, the methods encompass a method for regeneratingneural cells in a subject suffering from CNS damage includes applyingfunctional electronic stimulation (FES) to muscles of the subject thatare in communication with peripheral nervous system nerves of thesubject, the FES being sufficient to evoke patterned movement of limbsof the subject, so that the patterned movement in conjunction with theFES regenerates neural cells in the subject by promoting neural cellbirth and survival in the CNS of the subject.

In another embodiment, the methods encompass a method for repairing CNSdamage in a subject in need of such treatment includes regeneratingneural cells in or around a site of the CNS damage using functionalelectronic stimulation-evoked patterned movement.

In another embodiment, the methods encompass a method for at leastpartially restoring sensory or motor function in a subject sufferingfrom CNS damage, the method including inducing in the subject an amountof FES-evoked patterned movement sufficient to at least partiallyrestore motor or sensory function previously unattainable for thepatient due to the CNS damage.

In another embodiment, the methods encompass a method for at leastpartially restoring motor function in a motor complete subject includinginducing in the subject a therapeutically effective amount of FES-evokedpatterned movement.

In another embodiment, the methods encompass a method for at leastpartially restoring sensory function in a sensory complete subjectincluding inducing in the subject a therapeutically effective amount ofFES-evoked patterned movement.

In another embodiment, the methods encompass a method for at leastpartially restoring motor and sensory function in a subject who is bothmotor and sensory complete, including inducing in the subject atherapeutically effective amount of FES-evoked patterned movement.

In another embodiment, the methods encompass a method for treatingspinal cord injury including identifying a motor complete or sensorycomplete subject in need of such treatment, regenerating neural cells inthe spinal cord of the subject using functional electronicstimulation-evoked patterned movement, and monitoring of the subjectduring and after the course of said treatment to assess theeffectiveness of the neural cell regeneration.

In another embodiment, the methods encompass a method for late recoveryof sensory or motor function in a subject suffering from CNS damage,including inducing in the subject a therapeutically effective amount ofFES-evoked patterned movement to muscles the control of which has beenaffected by the CNS damage wherein the induction of FES-evoked patternedmovement is begun more than six months after the CNS damage occurs.

In another embodiment, the methods encompass a method for regeneratingneural cells in a subject suffering from CNS damage that includesapplying functional electronic stimulation (FES) to muscles of thesubject that are in communication with peripheral nervous system nervesof the subject, the FES being sufficient to stimulate a central patterngenerator coordinating patterned movement of limbs of the subject, sothat the stimulation of the central pattern generator regenerates neuralcells in the subject by promoting neural cell birth and survival in theCNS of the subject.

In another embodiment, the methods encompass a method for repairing CNSdamage in a subject in need of such treatment includes regeneratingneural cells in or around a site of the CNS damage FES stimulation of acentral pattern generator.

In another embodiment, the methods encompass a method for at leastpartially restoring sensory or motor function in a subject sufferingfrom CNS damage, the method including stimulating a central patterngenerator in the subject using FES configured to evoke a patternedmovement in the subject.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdescription, examples and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows x-ray films revealing an open mouth view (A) and a lateralview (B) of the cervical spine following internal stabilization in ahuman subject with SCI;

FIG. 2 shows schematic drawings of an FES bicycle;

FIG. 3 shows serial MR images of the cervical spinal cord demonstratinga posttraumatic cyst at C-2 level and severe encephalomalacia in thesubject, a human patient who suffered a traumatic SCI 5 years earlier;

FIG. 4 is a schematic illustration of the timeline of injury andcomplications of the human patient of FIG. 3;

FIG. 5 displays graphs describing marked recovery of the human patientfrom SCI;

FIG. 6 is a schematic drawing comparing the human patient's ASIA gradesfrom 1995 to 2002;

FIG. 7( a) schematically illustrates the methodology used in a rat modelof spinal cord injury to evaluate the effects of FES-evoked patternedmovement;

FIG. 7( b) is a time line of the experimental design for the rat modelof spinal cord injury;

FIG. 8 shows new cell birth in the adult spinal cord after chronic SCI;

FIG. 9( a) shows results of quantitative counts of BrdU labeled cellsshowing FES-induced selective increase in new cell birth reserved tolower lumbar segments;

FIG. 9( b) shows that the effect demonstrated in FIG. 8( a) persisted inthe cell survival group 7 days later;

FIG. 10, (a)-(l) shows confocal microscopic images of anti-BrdU labelledand phenotype labelled cells identifying new-born cells as tripotentialprogenitor cells, glial progenitor cells, astrocytes andoligodendrocytes, from an FES-treated animal at lumbar level L5, twohours after the last BrdU injection;

FIG. 11, (a)-(e) shows quantification of NG2 (glial progenitor cellmarker) colocalization with BrdU following spinal cord injury at 2 hoursafter last BrdU injection;

FIG. 12, (a)-(e) shows quantification of GFAP (astrocyte marker)colocalization with BrdU in the injured spinal cord at 2 hours afterlast BrdU injection;

FIGS. 13, (a)-(e) shows quantification of Nestin (tripotentialprogenitor cell marker) colocalization with BrdU in the injured spinalcord at 2 hours after last BrdU injection;

FIG. 14 show multiple views of a T1-weighted MRI of cervical SCI in theSCI subject of FIGS. 1-6;

FIG. 15 shows 3D and 2D flattened views of an atlas brain with projectedBOLD responses for visuomotor tracking task;

FIG. 16 shows 3D and 2D flattened views of the atlas brain in which BOLDresponses for sensory vibrotactile stimuli have been projected; and

FIG. 17 is a comparison of functional topography in primarysomatosensory cortex (SI), between the human subject with SCI andcontrol subjects.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To facilitate understanding of the invention, certain terms as usedherein are defined below as follows:

As used herein, the term “therapeutically effective” refers to acharacteristic of an amount of FES-evoked patterned movement, whereinthe amount is sufficient to at least partially restore previously lostmotor or sensory function or previously lost motor and sensory function,together with regeneration of neural cells in the subject. In subjectssuch as motor complete or sensory complete subjects, regeneration ofneural cells is deemed to occur when at least partial restoration ofpreviously lost function is observed. In other subjects where theexistence of undamaged, spared neurons may partially contribute torecovery of function, regeneration of neural cells can be evaluatedusing fMRI and other imaging techniques, or other methods of tissueevaluation as known in the art.

As used herein, the terms “regenerate” and “regeneration” refer to theenhanced birth and survival of neuroepithelial-derived cells of thecentral nervous system, including particularly cells of the spinal cord,within or surrounding a site of cell damage. Neural regeneration shallbe understood to at least partially restore or enhance neuronalsignaling through or around the site of cell damage.

As used herein, the term “neural cells” encompasses tripotentialprogenitor cells, glial progenitor cells, astrocytes andoligodendrocytes, or any combination thereof, as well as any other cellsthat may derive developmentally from the neuroepithelium.

As used interchangeably herein, the terms “central nervous systemdamage” and “CNS damage” refer to the result of a disease process orinjury that is characterized by destruction of, or harm to, cells of thebrain or the spinal cord, such that the normal motor and sensory controlfunction of the brain or spinal cord is disrupted. CNS damage shall beunderstood to encompass, for example, the result of an acute traumaticbreak or injury of the spine that completely or partially severs thespinal cord, the result of a stroke, the result of chronic disease suchas multiple sclerosis, Huntington's Disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS) and neurodegeneration of aging, andthe result of cancerous tumors forming within the central nervoussystem. A subject suffering from CNS damage is deemed to also besuffering from at least a partial disruption of motor or of sensoryfunction, or of both motor and sensory function as a result of the CNSdamage.

As used interchangeably herein, the terms “spinal cord injury” or “SCI”refer to a specific instance of CNS damage characterized by complete orpartial destruction of the spinal cord at one or more sites, which mayresult from acute trauma to the spine or from a disease process.

As used interchangeably herein, the terms “functional electricalstimulation” and “FES” refer to a form of electrotherapy in whichexternal electric current is applied to a muscle or muscle group tothereby evoke a bodily movement that normally would be subject tovoluntary or involuntary control by the CNS.

As used interchangeably herein, the terms “central pattern generator”and “spinal pattern generator” refer to a group of nerve cells in thecentral nervous system, particularly in the spinal cord, that interactin a circuit to produce a coordinated pattern of signals that stimulatemuscles to contract in sequence. An example is the alternating movementsof leg muscles during walking. Although all the nerve cells in thecentral pattern generator may be located in the spinal cord, theactivity of the circuit is normally modulated by signals from the brain,so that the activity is normally under voluntary control.

As used herein, the term “patterned movement” refers to motor activityof a subject, in which a body part or parts, typically a limb or limbssuch as the legs, follow a coherent and repetitive or cyclicaltrajectory. When more than one body part is involved in a patternedmovement, the body parts maintain a discernible coherentinterrelationship during the motor activity. Examples of patternedmovement include walking, breathing, biking, punching, kicking,swimming, fist clinching, toe pointing, knee bending, hip flexing,sitting, standing, and jumping.

As used herein, the term “FES-evoked” is used to described motoractivity of a subject that is partially or completely elicited byadministration of electrical stimulation to muscles of the body partsinvolved in the motor activity.

As used herein, the term “reciprocal movement” refers to a type ofpatterned movement in which at least two limbs, such as a pair of legs,move through at least two alternating positions in inverse relation toone another. Examples of reciprocal movement include the activity of twolegs in walking or in biking motion.

As used herein, the term “neural signal” refers to an electrochemicaldepolarization or hyperpolarization, or a combination thereof, of aneural cell, that transmits information between neural cells. A neuralsignal may be an action potential, consisting of a stereotyped sequenceof depolarization and repolarization of the cell membrane that transmitsinformation through the cell from the input of the cell to its output. Aneural signal alternatively be a local hyperpolarization ordepolarization of the cell membrane that renders the cell either more orless likely to fire and action potential.

As used herein, the term “subject” refers to a mammal, including humansand nonhuman primates, dogs, cats, horses, cows, sheep, rabbits, mice,rats, and guinea pigs.

The present invention is based in the surprising discovery that atreatment regimen including induction of FES sufficient to evokepatterned movement in a subject suffering from CNS damage, such as amotor complete subject, at least partially restores previously lost CNSfunction and regenerates neural cells in or around a site of CNS damage.Accordingly, the present invention provides a novel approach to at leastpartially restoring motor and sensory function in individuals sufferingfrom CNS damage, and particularly those individuals with such extensiveCNS damage that recovery of any function requiring the activity of thelost or damaged neurons was previously thought impossible.

The methods are based in part on surprising findings in a human subjectwith a high cervical (C2) traumatic injury, clinically assessed as motorcomplete. The patient showed only spotty sensation in the left hemibodyover the five years following the injury. When treated with a regimen ofphysical therapy that included application of functional electricalstimulation (FES) sufficient to evoke patterned movement in thesubject's lower limbs, the subject showed unexpected, late partialrecovery of motor function during years 6-8 post-injury. The methods arealso based in part on complementary experimental findings in a rat modelof spinal cord injury which, consistent with the human findings,demonstrate the surprising effects of the current methods on neural cellregeneration and on motor function in rat.

Conventional thinking has held that nerves in the adult mammalian CNSwill not and cannot be induced to regenerate. Thus, recovery of lost CNSfunction in a patient suffering from CNS damage has previously beenlimited to that function that can be recovered solely through retrainingexisting, undamaged (i.e. “spared”) neural cells. By “retraining” ismeant application of a therapeutic regimen such as locomotor trainingthat may result in improved function through recruitment of existing,undamaged motor neurons and from the induction of synaptic changes amongexisting neurons and between neuron and muscle fibers, such that theexisting, undamaged cells are more efficiently used.

In contrast, Applicants have discovered that inducing FES-evokedpatterned body movements regenerates neural cells such that CNS damagepreviously thought beyond repair is repaired, and function previouslythought permanently lost is at least partially restored. Without beingbound to a particular theory, the FES-evoked patterned movements arethought to stimulate neural regeneration by stimulating neural activityin a central pattern generator. Physiologic and metabolic demands placedon cells comprising the spinal circuit may activate cellular processesthat promote new neural cell birth and survival.

Accordingly, the methods of the present invention encompass a method fortreating CNS damage in a mammalian subject in need of such treatment,including inducing in the mammalian subject to a therapeuticallyeffective amount of FES-evoked patterned movement. The therapeuticallyeffective amount of FES-evoked patterned movement involves, in part,exposing the subject to the FES-evoked patterned movement for a periodof time sufficient to regenerate neural cells, including the neuralprogenitor cells known as tripotential progenitor cells, as well asglial progenitors, astrocytes and oligodendrocytes. The regeneration isa result of enhanced neural cell birth or survival, or both enhancedbirth and survival, as demonstrated by the anti-BrdU cell labelingresults described infra. Further, as demonstrated by the human findingsand corresponding rat data, FES-evoked patterned movement at leastpartially restores function by enhancing neural signal transmissioncapacity in or around a site of CNS damage, such as in or around aspinal cord lesion. The resultant at least partial restoration offunction is also consistent with synaptic changes in both new andpre-existing neural cells form new synaptic connections among the cellsin or around a site of CNS damage.

In an exemplary embodiment as described infra, the FES-evoked patternedmovement is biking movement of the legs of a human subject, and the FESis applied to at least one leg muscle or muscle group. However,depending on the patterned movement selected for therapy, the muscle ormuscle groups selected for induction of FES will be selected asappropriate from, for example, the gluteals, paraspinals, abdominals,wrist extensors, wrist flexors, deltoids, biceps, triceps, hamstrings,and quadriceps.

In general, the methods contemplate application of FES sufficient toevoke a patterned movement of limbs that results from a cycle of neuralactivity that is initiated in the peripheral nerves, propagates to thespinal cord, is followed by neural inactivity first in the peripheralnerves and then in the spinal cord, and then the cycle of neuralactivity is repeated. However, the actual elicitation of movement,particularly in a severely compromised patient such a motor completepatient several years post-injury, is not required to obtain neuralregeneration. FES sufficient to stimulate a central pattern generatorthat coordinates the patterned movement, will produce the same effect.Thus, the methods also contemplate patterned electrical activation of acentral pattern generator, regardless of whether movement is actuallyeffected, to regenerate neural cells and at least partially restorefunction. This embodiment is especially suitable for treating severelycompromised patients, such as the human subject of Example 1, whosemuscle strength has been severely compromised by atrophy over months andyears post-injury. Initially, partial restoration of function isobtained by regenerating neural cells using FES induced activation of acentral pattern generator. Later, as improvement of function permits,continued and further improvements are obtained by the FES-inducedactivation of the central pattern generator which also effects actualmovement.

Stimulation protocols are generally known in the arts of physicaltherapy and rehabilitation and are described for example in G. M.Yarkony et al., Arch. Phys. Med. Rehabil. 73:78-86 (1992). However, inan exemplary embodiment, a therapeutically effective amount ofFES-evoked patterned movement is attained by inducing FES sufficient toevoke patterned movement for at least about one hour per day and atleast three times per week. An example of an effective level ofelectronic stimulation within the meaning of the present invention iselectronic stimulation delivered at 3 V, and using 200 μs monophasicpulses delivered at 20 Hz, as described in Examples 1 and 2. The FES maybe induced through externally placed electrodes and connectors, i.e. onthe skin, or through internally placed electrodes, such as implantedelectrodes.

The effects of FES are assessed by clinical assessment of ASIA grade,and by the use of other clinical criteria and imaging techniques asknown in the art that indicate regeneration of neural cells in or aroundthe site of CNS damage. In a clinically complete patient, for example,partial recovery of function that corresponds to an improvement of atleast one ASIA grade, with respect to motor or to sensory function, isindicative that regeneration has occurred. In patients assessed asclinically incomplete, the effects of FES are assessed using acombination of clinical criteria including improvement of at least oneASIA grade, time elapsed from the original injury, and as needed,imaging techniques such as fMRI that reveal neural regeneration. Forexample, FES is deemed to have regenerated neural cells in a patientclinically assessed as motor incomplete who has previously shown nosignificant improvement for about 6 months post-injury, but then showssubstantial improvement of function corresponding to at least one ASIAgrade after being exposed to FES in accordance with the methodsdescribed. FES is deemed to have regenerated neural cells in a motorincomplete patient or a sensory incomplete patient in whom thepreviously lost motor or sensory function is at least partially restoredto a level corresponding to an improvement of at least one ASIA grade,and wherein the restored function was previously unattainable solelythrough retraining existing, undamaged (i.e. “spared”) neural cells. Theassessment of FES effects can be supplemented with the use of fMRI orother imaging techniques such as those relying on paramagnetic tracersto further reveal and characterize the neural regeneration.

In another embodiment, the methods encompass a method for regeneratingneural cells in a subject suffering from CNS damage, the methodincluding applying FES to muscles of the subject that are incommunication with peripheral nervous system nerves of the subject, theFES being sufficient to evoke patterned movement of limbs of thesubject, wherein the patterned movement in conjunction with the FES ofthe peripheral nervous system nerves regenerates neural cells in thesubject by promoting neural cell birth and survival in the CNS of thesubject. The methods encompass treating a subject who is either motorcomplete, or sensory complete, or both. The methods also encompasstreating a subject who is motor incomplete or sensory incomplete or bothwherein such a patient is characterized by CNS damage that cannot berecovered by retraining of existing, undamaged neurons.

The regeneration of neural cells and resulting at least partialrestoration of motor function or sensory function or of both motor andsensory function involves restored transmission of a neural signal. Inthe case of a spinal cord lesion, for example, a partial restoration offunction involves an at least partially restored neural signal that cancross the site of spinal cord injury. The at least partially restoredneural signal can then be used by the subject to initiate voluntarycontrol, including voluntary motor control for example, of a musclepreviously denervated as a result of spinal cord lesion or as a resultof other CNS damage.

In another embodiment, the methods contemplate a method for treating CNSdamage in a subject in need of such treatment, the methods includingregenerating neural cells in or around a site of CNS damage usingfunctional electronic stimulation-evoked movement.

Alternatively, the methods encompasses at least partially restoringsensory or motor function in a subject suffering from CNS damage byinducing in the subject an amount of FES-evoked patterned movementsufficient to at least partially restore motor or sensory functionpreviously unattainable for the patient due to the CNS damage. By“unattainable” is meant CNS function that cannot be recovered solelythrough retraining existing, undamaged (i.e. “spared”) neural cells.

Another embodiment contemplates a method for treating spinal cord injuryincluding identifying a motor complete or sensory complete subject inneed of such treatment, regenerating neural cells in the spinal cord ofthe subject using functional electronic stimulation-evoked patternedmovement, and monitoring the subject during and after the course of saidtreatment to assess the effectiveness of the neural cell regeneration.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following specific examples are offered by wayof illustration only and not by way of limiting the remainingdisclosure.

Example 1 Neural Regeneration in Humans

Spinal cord injury (SCI) is a well-known cause of CNS damage resultingin substantial or total loss of CNS function, including loss of sensoryor of motor function, or both depending on the precise nature of theinjury. Use of the inventive therapy for late recovery from SCI injurymay result in substantial if not complete restoration of motor andsensory function, even with a stable ASIA Grade A injury. Such partialrestoration of function is possible even when the late recovery is wellbeyond the 6-months-from-injury time frame previously accepted as theperiod within which large improvements are possible for ASIA Grade Apatients. According to the methods of the present invention, laterecovery of function is possible even several years after injury.

This prospective, single-case study evaluated the potential forfunctional recovery from chronic spinal cord injury (SCI). The resultsare described fully in McDonald et al, J. Neurosurg. (Spine 2) 97:252-65(2002), which is herein incorporated by reference in its entirety,together with the primary references contained therein. The patient wasmotor complete with minimal and transient sensory perception in the lefthemibody. His condition was classified as C-2 American Spinal InjuryAssociation (ASIA) Grade A and he had experienced no substantialrecovery in the first 5 years after traumatic SCI. Clinical experienceand evidence from the scientific literature suggest that furtherrecovery would not take place. When the study began in 1999, the patientwas tetraplegic and unable to breathe without assisted ventilation; hiscondition classification persisted as C-2 ASIA Grade A.

FIG. 1 shows x-ray films revealing an open mouth view (A) and a lateralview (B) of the cervical spine following internal stabilization. Thetype of injury incurred by this patient leads to bone dissociation ofthe head and spine. Reconstruction required fusion of the occiput to C-2with titanium rods, wire, and bone graft.

Magnetic resonance imaging revealed severe injury at the C-2 level thathad left a central fluid-filled cyst surrounded by a narrow donut-likerim of white matter. Five years after the injury a program known as“activity-based recovery” was instituted. The hypothesis was thatpatterned neural activity might stimulate the central nervous system tobecome more functional, as it does during development. Specifically,FES-evoked bicycling movement was induced in the subject, three timesweekly, for about one hour per session.

FIG. 2 shows schematic drawings of the FES bicycle. The FES bicycle usescomputer-controlled electrodes to stimulate the legs muscles in specificpatterns. A paralyzed individual can therefore rotate the bicycle wheelseven though he is unable to control his leg muscles voluntarily. In thisstudy, three muscle groups (red) were stimulated bilaterally: thegluteal, quadriceps, and hamstring muscles. Electrodes (blue) went topads attached to the skin over each muscle: two pads for each quadriceps(1) and one for each hamstring (2) and gluteal muscle (3).

Over a 3-year period (5-8 years after injury), the patient's conditionimproved from ASIA Grade A to ASIA Grade C, an improvement of two ASIAgrades. Motor scores improved from 0/100 to 20/100, and sensory scoresrose from 5-7/112 to 58-77/112. Using electromyography, the inventorsdocumented voluntary control over important muscle groups, including theright hemidiaphragm (C3-5), extensor carpi radialis (C-6), and vastusmedialis (L2-4). Reversal of osteoporosis and an increase in muscle masswas associated with this recovery. Moreover, spasticity decreased, theincidence of medical complications fell dramatically, and the incidenceof infections and use of antibiotic medications was reduced by over 90%.These improvements occurred despite the fact that less than 25 mm²tissue (about 25%) of the outer cord (presumably white matter) hadsurvived at the injury level.

History: This 42-year-old, right-handed man sustained a displaced C-2Type II odontoid fracture due to an equestrian accident on May 27, 1995.The mechanism of injury was direct axial loading. The patient's horsestopped suddenly; the patient's hands were caught in the reins and his 6ft 4 in, 230-lb body was projected over the horse's head. He landeddirectly on the helmet in a near-perpendicular position. He was renderedapneic but was maintained at the scene immediately with artificialrespiration. He was transferred to the University of Virginia Hospital.

The Injury: FIG. 3 depicts serial T2-weighted MR images obtained in thepatient's cervical spinal cord 5 years after the C-2 SCI. FIG. 3 shows aposttraumatic cyst at C-2 level and severe encephalomalacia in thepatient who had suffered a traumatic SCI 5 years earlier. A sagittal MRimage is shown on the left (A) and the corresponding coronal sectionsare shown the right (B-E). For ease of identification, the perimeter ofthe cord is circled in black and the internal cyst is white in panels Cand E, which are duplicate images of panels B and D. Graph showingquantitative analysis of spinal cord area by using MR imaging signal.Areas (in mm²) of the MR image signal in cross sections through thespinal cord are shown as a function of distance from the cerebellartonsils (0 reference point). The lesion epicenter is indicated by thelowest point on the U-shaped area curve. Based on a normal C-2 cervicalcross-sectional area of approximately 1 cm², then approximately 25% ofthe MR imaging signal remains at the injury epicenter, representing adonut-like rim of tissue. FIG. 4 is a schematic illustration of thetimeline of injury and complications of the human patient.

The injury epicenter contains a central cyst on level with the lowerpart of the C-2 vertebral body. For ease of reference, the central cystis shown in white in insets C and E. In the same insets, black linesoutline the cord. As is typical in high cervical injuries, severeencephalomalacia (shrinkage) has almost halved the diameter of the uppercord. Despite the severe damage, cross sections through the lesion(horizontal lines with arrows pointing to insets B-E) indicate avariable donut-like rim of remaining white matter tissue. The presenceof a variable donut-like rim of tissue at the injury site is typical ofmost SCIs. Furthermore, the majority of clinically important motor andsensory tracts are normally present in the outer rim of white matter;however, the MR images do not indicate whether the donut-like tissue isfunctional cord or simply scar tissue. An interesting feature of thepatient's images is that the cystic area is confined to the C-2 levelrather than extending one level above and below, as is more typical withtraumatic SCI. The likely explanation is that the cervical canal iswider at C-2, which protects the cord more than the canal at otherlevels.

Quantitative measures of remaining spinal cord cross-sectional areas inthe upper cervical region revealed that approximately 25 mm² of tissueremained at the lower C-2 lesion epicenter, representing a donut-liketissue rim (FIG. 3F). The lesion epicenter was identified by reducedaverage signal intensities with corresponding minimal area values. Assuggested in the MR images, the area of the most severe injury wasprimarily confined to one level (labeled C-2 in FIG. 3F); however,substantial atrophy of the cord was also present rostral and caudal tothe injury level. This atrophy extends further rostral than caudal inkeeping with the greater rostral axonal dieback observed in highcervical lesions in rodents.

Examination: Examination was consistent with a complete motor andsensory quadriplegia. Cervical traction was instituted. He receivedmethylprednisolone (30 mg/kg bolus, followed in 1 hour by 5.4 mg/kg)after the injury. Cervical spine x-ray films demonstrated a Type IIodontoid fracture with fracture of the occipital condyle anddisplacement of the occiput anterior to C-1, suggesting occipitoatlantaldislocation.

The current case reinforces the data noted above that limited sparing ofwhite matter may be associated with substantial preservation of motorand sensory function. In the patient's case, high-resolution MR imagingdemonstrated that less than 25% of the cord had survived at the injurylevel (and the proportion was probably lower because MR imaging cannotdistinguish between functional tissue and scar tissue). Yet substantialmotor and sensory recovery was possible. These observations imply thatsmall stepwise treatments can be expected to produce large gains infunction. Development and application of novel methods for determiningpersistent connections across the lesion in individuals with ASIA GradeA injuries will be important. Such techniques could include diffusiontensor imaging, functional MR imaging, and motor/sensory evokedpotentials.

The MR imaging analysis of the lesion indicated residual sparing ofapproximately 25 mm² of tissue in an outer donut-like rim of the cord atthe C-2 level. The analysis would tend to err on the side of inflatedarea measurements, as the demarcation of tissue and cerebrospinal fluidin the central cyst was not always a clean border. Nonetheless, thelesion epicenter can be identified in FIG. 3 by a simultaneous drop insignal intensity and minimal residual area. Previous work in normalvolunteers suggests the area of C2-4 spinal cord cross-sections rangesfrom 67 to 101 mm², with study means ranging from 78.1 to 84.7 mm².Previously published data obtained in normal human volunteers at theC2-3 and C3-4 disc space levels, based on MR imaging, is as follows:mean age 31 years (range 23-49 years), area 78.1 mm² (range 70.1-86.1mm²) for 15 volunteers; mean age 38 years (range 26-57 years), area 84.7mm² (range 67-101 mm²) for 30 volunteers. Therefore, it is reasonable toassume that approximately 25% of the cord remains at the injury level inthe patient.

Operation and Immediate Postoperative Course: Nine days later occiput-C2fusion was performed (FIG. 1) using a titanium ring and sublaminar softwire at C-1 and C-2. Bone was obtained from the iliac crest. Because theMR imaging appearance was consistent with preserved neural tissue, greatcare was taken to maintain alignment during the procedure and inparticular when turning the patient from supine to prone.

As is usual in high cervical injuries, the patient underwenttracheostomy and gastric tube placement. All the early ASIA examinationsfor SCI demonstrated similar results. For example, the examination onJun. 24, 1995, almost 1 month after the injury, indicated aclassification of ASIA Grade A, with complete motor and sensory functionat level C-2, partial preservation of sensory function at C-3, andspotty sensation in the left hemibody, this level including absence ofsacral function such as voluntary anal contraction. The patient receiveda single test dose of GM-1 ganglioside 39 days after injury, butmastocytosis precluded further doses. The patient's initial hospitalcourse, including inpatient rehabilitation, was uncomplicated except bysacral skin breakdown (Grade IV that healed by secondary intention).When the patient was discharged from the hospital on Dec. 13, 1995,approximately 5 months after the injury, he was dependent on aventilator and his SCI was classified as C-2 ASIA Grade A.

Long-Term Treatment Course:

1. Informed Consent and Approval. The Human Studies Committee atWashington University in St. Louis approved the methods for collectingdata and presenting the case. Informed consent was obtained forparticipation in research activities in accordance with this committee'sstandards from the individual described herein. Inclusion of the qualityof life questions and answers were approved by the patient.

2. The “N of 1” Study Design. The “N of 1” analytical method is anaccepted rational design that has attracted interest from the NationalInstitutes of Health.

3. The ASIA Examinations. The ASIA examinations spanning the periodprior to recovery (1999) through recovery (end 1999-2002) were performedin accordance with ASIA's International Standards by a singleinvestigator (J.W.M.) trained in the measurements and in the specialtyof SCI medicine. The results of early examinations were obtained fromASIA sheets in the original hospital charts (1995-1999). The individualswho performed these examinations were also experts in SCI care and wellversed in the clinical ASIA standard examination. Initial examinationsby the serial examiner (J.W.M.) confirmed these earlier records (1999).All components of the examination were performed in accordance with ASIAstandards while the individual was lying in bed. The ASIA InternationalStandard test for patients with complete tetraplegia has good intra- andinterrater reliability.

4. Electromyography Evaluation. The Advantage/Clarke-Davis (diaphragm)and Dantec/Keypoint (upper and lower extremity muscles and externalsphincter muscle) instruments and a Medtronic disposable monopolarneedle DMN 50 were used for electromyography. Amplifier input impedancewas set at 5 kOhm, with a high-pass filter of 2 Hz, a low-pass filter of10 kHz, a sweep of 10 to 200 msec/D and a sensitivity of 0.1 to 0.2mV/D. The diaphragm was evaluated while the patient was seated, and thefollowing muscles were assessed while the patient was lying down: rightdeltoid, biceps, extensor carpi radialis, and vastus medialis.

5. Bone Density. Bone densitometry was performed at WashingtonUniversity School of Medicine's Bone and Mineral Diseases laboratories.Dual-energy x-ray absorptiometry measurements were compared usingnational standards for white males (based on age, weight, and height).Data are presented as standard deviations using t-scores (sex-matchednormal young adult reference).

6. Magnetic Resonance Imaging

Safety Testing:

Before placing the patient in the MRI scanner, duplicate copies of theorthopedic instrumentation were acquired for testing of MRcompatibility. To simulate the titanium loop instrumentation in thepatient's cervical region (Danek Inc., Minneapolis, Minn.), twostainless-steel Harrington rods and stainless steel circlage wires wereobtained for testing. A neurosurgeon bent the rods using a standardbending apparatus to approximate the bend of the straight segments ofthe patient's Danek loop, using the patient's plain-film radiographs.This bending was performed because stainless steel could become slightlymagnetic when bent.

The rods and wires were manually passed in and out of the MRI scanner.No torques were felt on the metal that would indicate any force due tomagnetic properties. A strong hand-held magnet was also moved around themetal, and no attractive forces were felt. The rods and wires were thenarranged to match the configuration on the radiographs. The hardware wastaped to a spherical phantom containing 0.9% NaCl (saline) and dopedwith 0.15 mM Gd[DTPA-BMEA] (Optimark, Mallinckrodt Inc., St. Louis, Mo.)to shorten the T1 relaxation time to the physiological range. Thephantom was placed in the MRI scanner, and several scans were performedusing various pulse sequences that were planned to use in subsequentpatient imaging sessions (MP-RAGE, turbo spin echo, echo-planar imaging,diffusion tensor imaging, etc.). The investigator held his hand over themetal during the imaging, and did not feel any RF heating or movement ofthe metal.

Patient setup: The patient was transported to the MRI suite by van, andall external metal was removed from his pockets. During transport andMRI the patient was ventilated using his personal ventilator connectedto a tracheostomy tube for positive ventilator assistance. Outside theMRI room, the patient and his ventilator were moved onto a lightweightaluminum stretcher (Model 30NM, Ferno-Washington Inc., Wilmington, Ohio)tested for MR-compatibility, and then the patient was transported intothe MRI room. A pulse oximeter probe was placed on his right indexfinger to monitor heart rate and oxygen saturation (using a rubber gripsensor, Invivo Research, Inc., Orlando, Fla.). A non-invasive bloodpressure cuff was placed over the left arm and a tubing was connected toa side-port of the ventilator connector to monitor blood pressure,respiratory rate, end-tidal CO₂, and inspired O2 (3150 Magnitude/3155AMillenium anesthesia monitoring system, Invivo Research, Inc., Orland,Fla.). The ventilator tubing was temporarily disconnected and thepatient was lifted onto the MRI table. The ventilator tubing wasimmediately reconnected, and a wall O₂ line was connected to theventilator (medical grade O₂, Airgas, St. Louis, Mo.). The oxygen flowwas adjusted to 2-3 L/minute to maintain SpO2 to >95% in the supineposition. (In the upright position the patient typically has an SpO₂ of97-98% without additional O₂.) The patient was positioned so that theC2-C3 region of the cervical spin was centered over the active elementof the neck phased array coil (see below). Padding was placed under theelbows, knees, and back. The ventilator and O₂ line were temporarilydisconnected and moved to inside a non-magnetic RF-shielded box(Shielding Resources Group, Tulsa, Okla.) placed next to the MRIscanner. The RF box contained a shielded AC/DC power source, shieldedelectronic connectors, and waveguides (copper conduits) built into thewalls of the box. The ventilator and O₂ tubings were fed through awaveguide and reconnected to the ventilator inside the RF box. This RFbox was tested at >80 dB attenuation to completely screen RF emissionsfrom the ventilator and remove severe RF interference in the MRI.Headphones were placed on the patient for acoustic shielding andlistening to music, and tape was placed across the forehead to minimizemovement of the head and neck. The patient was then advanced into thescanner by moving the patient table, positioning the C2-C3 region andactive coil element at the isocenter of the magnet and gradients. Thedisplay from the Invivo monitor was then connected from a custom videoport of the Millenium 3155A anesthesia unit through a custom RFpenetration panel in the MRI room wall (Shielding Resources Group,Tulsa, Okla.) and then to the back of the satellite MR console monitorfor display of physiologic data in the control room, where aneurointensivist monitored all physiologic parameters during MRI. Duringthe entire setup and imaging period, the heart rate was 58-86 beats perminute, SpO₂ was 95-97%, blood pressure was 126-139 mm/72-86 mm, therespiratory rate was 18 respirations per minute (equal to the ventilatorsetting), the end-tidal CO₂ was 29-34 mm Hg, and the inspired O₂ (FiO2)was 28%. The patient did not experience any adverse symptoms during theimaging sessions.

MRI Acquisitions: A 1.5-Tesla Siemens Magnetom Vision MRI scanner andSiemens phased-array spine coil were used for all imaging (Siemens,Erlangen, Germany). After an initial scout to confirm positioning at theisocenter, the external magnetic field was shimmed manually using thevendor MAP-shim procedure. For this purpose, only the linear shimchannels were used because of instability in the higher-order shimchannels due to the adjacent metal instrumentation. The frequency andtransmitter were then tuned to the on-resonant and 180° conditions, andthe scout scan was repeated. A T1-weighted MP-RAGE scan was acquirednext in the transverse plane, tilted ˜20° sagittally and coronally to beperpendicular to the cord. The scan had 1.0-mm isotropic voxels (256×256mm field of view and 256×256 matrix, 160-mm z-slab with 160z-partitions) with TE=4.0 ms, TR=9.7 ms, TI (inversion time)=472 ms, anda 12° tip angle. The acquisition time was 9 min 29 sec with no signalaveraging. The MP-RAGE data was used for cord cross-sectional areameasurements (see below). Additional anatomical scans such asT2-weighted turbo spin echo and fluid-attenuated turbo inversionrecovery were also acquired.

Data analysis: MP-RAGE data were resliced to be perpendicular to thecervical cord. Region-of-interest (ROI) tracings were made around theborder of the cord on every other transverse image using the “autotrace” function of Analyze AVW 4.0 (Mayo Foundation, Rochester, Minn.).To choose the intensity level for the auto-trace, the lower level wasdetermined in which the trace expanded out into the surroundingbackground noise, the higher level was determined in which the tracecollapsed into the center of the cord. Then, the mean of the two levelswas calculated and chosen for the auto-trace of the cord area. By thisprocedure, partial volume effects were taken into account in which thecord intensity transitions from high T1-weighted intensity to the lowsignal surrounding the cord (see FIG. 3). In some slices, the ROIgenerated by the auto-trace procedure had to be edited to prevent theregion from extending into adjacent tissues (e.g., due to cordadhesions). The two-dimensional areas of the cord were measured bysampling the ROIs, and results were graphed as a function of lengthalong the cord.

7. Quantification of Infectious Complications. The personal 24-hournursing records for the patient were exceptionally detailed and thisallowed us to track accurately the number and types of infectionsrequiring antibiotic treatments each year. In addition, the totalduration of each treatment was always recorded. The prescription recordsprovided by the local medical doctor verified these values. In mostcases, cultures were also obtained to further verify the infection.

8. Quality of Life Evaluations. A single examiner (J.W.M.) performed theevaluations by telephone (Jul. 15-30, 2002). These subjectivemeasurements supplemented the quantitative data on functional recoveryand emphasized the impact of limited motor and sensory recovery onquality of life.

9. Activity-Based Recovery Program. The activity-based recovery programconsisted primarily of training on a FES bicycle. The customizedrecumbent bike system, designed for use with paralyzed individuals,integrated computer-assisted FES-induced cycling. The goal was 1 hour ofactivity (up to 3000 revolutions) per day three times per week. The FESbicycle modulates the intensity of stimulation to obtain a consistentrotation speed. Surface electrodes stimulate three muscle groups in eachleg (FIG. 2): one electrode is placed at the superior edge of thegluteal muscle, another over the hamstring group midway between the kneeand hip, and two over the quadriceps (one over the superior portion andthe other over the inferior third of the quadriceps). During theexercise, the legs were balanced in three ways. The seated buttocks andboots anchored the legs at the upper and lower positions. Belts thatattached to the upper leg with Velcro balanced the mid-leg. A weightedfly-wheel ensured a smooth rotation by carrying momentum. The goal wasto achieve the greatest number of revolutions (3000/hour). The FESbicycle therapy was supplemented with surface electrical stimulation toactivate the following muscle groups; paraspinals, abdominals, wristextensors, wrist flexors, deltoids, biceps, and triceps. The therapieswere rotated daily, usually in a 3-day sequence. Each muscle group wasactivated for half an hour using intermittent 1 second on, 1 second offAC cycles. Once muscle recovery began, aquatherapy was incorporated intothe program, with a goal of one 1-hour session per week. The aquatherapyfocused on muscle groups in which voluntary control was recovered whileparticipating in the activity based recovery program.

Upon first evaluating the patient, the activity-based recovery program'seffects on physical as well as functional recovery was studied. Earlyresults suggested that the physical benefits alone were sufficientreason to incorporate activity-based therapy into daily life. Thosebenefits included enhancement of muscle mass and bone density, increasedcardiovascular endurance, and decreased spasticity (data not shown).

After the initial evaluation at Washington University in St. Louis, thepatient was trained to use a FES bicycle (FIG. 2). A similar bicycle wasinstalled in his home so he could exercise frequently in his city ofresidence. The goal was to complete a 1-hour session three times perweek.

At first, the patient's leg muscles fatigued rapidly with surfacestimulation, but within approximately 20 sessions he was able to ridethe bicycle continuously for 1 hour. Once motor recovery began, theprogram was supplemented with weekly aquatherapy to work muscle groupsthat had regained voluntary function but were too weak to opposegravity. Surface, non-load bearing electrical stimulation was alsoperformed on the following muscle groups on an alternating 3-dayschedule: the paraspinal group, the abdominal group, and theupper-extremity groups. Standard range-of-motion physical therapy wasalso performed daily, but this regimen was not changed from the time theindividual was first discharged from rehabilitation. Breathing exercisesalso became part of the daily routine beginning in 1998. Increasedmuscle size and a generalized improvement in health were clearly evidentby the year 2000; however functional recovery was slower.

1. Major Medical Complications. As usually happens with a high cervicalSCI, the patient developed many severe medical complications,particularly between 1996 and 1999 (FIG. 4). The patient suffered a C-2ASIA Grade A SCI on May 27, 1995. As often happens in tetraplegia, heaccumulated many severe medical complications, which are listed in thistimeline. The vertical red bar indicates inpatient medical care andrehabilitation through the end of 1996. In addition to the complicationsshown here, urinary tract and pulmonary infections were frequent in theyears before 1999. Note that the complication rate accelerated between1995 and 1999, a situation common in tetraplegia. The paucity of similarcomplications after 1999 is highly unusual. 1) Coccyx skin ulcer. Alarge, Grade IV sacral skin ulceration developed early during thehospitalization period. Aggressive treatment produced healing bysecondary intention 1 year later (the vertical green bar indicateshealing time). 2) HO. Heterotopic ossification (lesser trochanter ofright femur) developed in July 1995, as indicated by acute swelling ofthe right thigh. A Doppler study was negative for deep venousthrombosis, and x-ray films and CT scanning failed to reveal a fracture.Treatment with Didronel was initiated to prevent further boneresorption. Heterotopic ossification is a common complication in theacute postinjury period. 3) AD. Severe autonomic dysreflexia requiredinpatient treatment. 4 & 5) DVTs. Left deep venous thrombosis occludedthe large draining vein of the leg and required hospitalization foranticoagulation with heparin and then Coumadin. The DVT recurred 1 monthlater, requiring adjustment of the anticoagulation regimen and placementof an inferior vena cava (IVC) Greenfield filter. Life-long treatmentwith Coumadin and weekly blood anticoagulation testing was thenrequired. 6) Pathological fracture. Pathological fracture of the leftfemur resulted from a low fall during transfer. Surgical interventionwas required to stabilize the fractured bone. 7) Collapsed lung. Acuteshortness of breath required emergency hospitalization and bronchoscopyto remove a mucous plug. 8) Ankle ulcer. A left lateral malleolus skinulceration (Grade IV) was complicated by slow healing and osteomyelitis,threatening amputation. Aggressive treatment and healing by secondaryintention took more than 1 year. 9) Sacral skin ulcer. Pilonidal cystremoval and suture closure was complicated by dehiscence and developmentof a sacral wound that required aggressive treatment and healing bysecondary intention (vertical green bar indicates healing time). 10)Pathological fracture. The left femur fractured while the patient wasattempting weight-supported standing. Treatment required hospitalizationand surgical internal fixation. Additional treatment of severeosteoporosis included vitamin D, calcium supplements, andpharmacological treatment to limit bone resorption.

Despite his severe injury and immobility, however, he fared extremelywell. These complications included skin breakdown, heterotopicossification, autonomic dysreflexia, pathological bone fractures, deepvenous thrombosis, and acute respiratory distress consequent to mucousplugging.

2. Recovery of Function. The patient experienced no functionalimprovement during the first 5 years after injury, thereby retaining theC-2 ASIA Grade A classification, and the likelihood of any substantialimprovement in the following years seemed negligible. Multiple ASIAexaminations during the first 5 years after the patient's injurydocumented a lack of substantial motor or sensory function because motorscores were consistently 0/100 and sensory scores ranged from 5-7/112.The first ASIA examination at Washington University in 1999 wasconsistent with these observations. FIG. 5 shows graphs quantifying themarked recovery from SCI. Data are presented as parameter values(x-axis) and total motor and sensory function (light touch, pinprick;y-axis) based on the international ASIA standard scale for ratingseverity of SCI.

Motor function was assessed in five myotomes in the arms and fivemyotomes in the legs, and the scale of 0 to 5 provided a maximum scoreof 100 (10 muscles on each side). Sensation was measured in 28dermatomes on each side. The scale of 0 to 2 (0=absent, 1=impaired,2=normal) provided a maximum score of 112. The ASIA classification runsfrom Grade A to E, with E being normal. This individual displayed nomotor function during the first 5 years after injury. Sensory functionover the first 5 years was restricted to upper neck dermatomes. Theactivity-based recovery program was instituted mid-1999. After 6 months,no recovery was observed; however, progressive and considerable motorand sensory recovery was evident over the next 2.5 years.

The patient did begin to recover sensation to deep palpation in theupper torso and upper arms in early 1999, but this did not translateinto substantial changes in the ASIA grade because since detection oflight touch and pinprick sensation are the only measures.

After the first 6 months of the activity-based recovery program (January2000), little recovery of function was evident: the arms and legs stilldisplayed no motor function and a light touch or pinprick could besensed only above the affected dermatomes in the neck (C1-C2/3). Thepatient first noted the ability to control a twitching movement of hisleft index finger in early November 2000. The first documented recoveryon ASIA examination was at the end of 2000 (FIG. 5). Although modestimprovements in sensory and motor scores were observed, sacral sparingof light touch sensation was first evident, changing the patient's ASIAclassification to ASIA Grade B. Twenty-two months into the program (July2001), however, light touch sensation improved to 52% of normal. Itrecovered to 66% of normal in 2002. Pinprick assessment, which requiresdiscrimination and is therefore more difficult, did not improve untilthe last year of the 3-year program, which is ongoing to date. Sensationwas appreciable throughout most dermatomes of the body (FIG. 6),including the sacral region (S-3, S4-5). In addition to recovery ofpinprick and light touch, recovery of additional sensory modalitiesoccurred and included vibration, proprioception, and ability todifferentiate heat and cold.

The most notable change was an improvement in motor function of up to20% (20/100). Voluntary control of the external anal sphincter ispossible (S4-5). The conversion of the patient's condition to ASIA GradeC thus occurred in July 2001. Motor recovery was first evident in theleft fingers, then the right hand, and then the legs. Movement is nowpossible for most muscles of the upper arms but for fewer muscles in thelegs. Most muscles in the legs are not yet able to oppose gravity. FIG.6 is a schematic drawing showing a comparison of the patient's ASIAgrades from 1995 to 2002. Values in blue indicate scores from 1999;those in red indicate 2002 scores. Note that all the 1995 motor scoresand the sensory scores obtained below T-4 were zero. The 1995 scoreswere taken from the July 1995 ASIA sheet.

The following comparison puts this motor recovery into perspective. TheNational Acute Spinal Cord Injury Study II and III trials documented anaverage 4.8-point improvement in the motor scores of patients receivingmethylprednisolone compared with those receiving placebo ifmethylprednisolone was given within 8 hours of injury.

In the patient's case, there was a 20-point improvement in motor scoreover Years 5 to 8 post-injury (FIG. 5), which translates into movementin most joints, including the elbows, wrist, fingers, hips, and knees(FIG. 6); thus, the patient's condition is now categorized as ASIA GradeC rather than Grade A. This degree of movement gives the patient bettercontrol of the environment and of his powered wheelchair. The directconsequences of the recovered movement are limited but the consequencesof the sensory recovery are substantial. In addition, the other benefitsassociated with enhanced muscle function have tremendously improved thepatient's quality of life.

3. Reduction of Infections and Physical Improvements. Strikingly, thepatient's infection complication rate began to decline dramatically in1999, in parallel to reductions in major complications and recovery ofneurological improvements. The incidence of infections requiringantibiotic treatment and total days of antibiotics per year alsodramatically improved following 1999. Evaluation of total days ofantibiotics required revealed over a 90% reduction in Years 2000 to 2002compared with Years 1996 to 1998 (Table 2).

TABLE 2 Yearly Incidence of Infections and Requirement for AntibioticTreatment Total events Total days requiring Urinary Skin of antibiotictract Pulmonary Bowel infec- antibiotic Year treatment infectionsinfections infections tions treatment 1996 23 10 9 3 1 169 1997 13 4 4 04 190 1998 13 5 6 0 2 168 1999 8 1 4 0 3 99 2000 5 3 2 0 0 36 2001 3 3 00 0 18 2002 1 1 0 0 0 10

Events were recorded from detailed personal nursing records and indicatetotal number of infectious events requiring antibiotic treatment, typesof infections and total days of antibiotic treatment required.

In addition to these improvements, the patient also achieved substantialphysical benefits. His severe osteoporosis, which contributed topathological fractures of two of the largest bones in his body (femurand humerus), was completely reversed and is now within the normal range(bone density t-score−0.5 in 2002 compared with −4.1 pre-1999). Ashworthmeasurements of spasticity have improved from 3 to 1-2 (Table 3), andthe patient has also increased his endurance.

TABLE 3 Ashworth Scale for Measuring Spasticity Ashworth Score Degree ofMuscle Tone 1 No increase in tone 2 Slight increase in tone resulting ina “catch” when affected limb is moved in flexion and extension 3 Moremarked increase in tone; passive movement difficult 4 Considerableincrease in tone; passive movement difficult 5 Affected part rigid inflexion and extension

4. Electromyography Results. The EMG analysis of volitional movementswas completed in the winter of 2001 (Table 4), and the results werecompared with those from phrenic nerve testing performed on Jun. 21,1995, shortly after the injury. At that time, there was evidence ofintact anterior horn cells. Latencies were less than 10 msec, and rightand left amplitudes were 0.9 mV and 0.5 mV, respectively. Diaphragmaticmovement, albeit small, was noted on fluoroscopic examination. Incontrast, amplitudes were greater in 2001 (2-7 mV), but there wasfurther evidence of denervation (Table 4). Voluntary elicited EMGresponses were evident in other muscle groups tested, including theright deltoid, right biceps, right extensor carpi radialis, and rightvastus medialis. Most of these groups showed evidence of denervation, asindicated by positive sharp waves, fibrillation, and complex repetitivedischarges. Overall, the numbers of recruited motor units werepredictably small.

TABLE 4 EMG Characteristics of Muscles During Volitional ActivationSpontaneous Response # Motor activity Motor unit characteristicsVoluntary latency units Insertional (PSW, Fibs, Amplitude DurationMuscle group response (ms) recruited activity CRDs) (mV) (ms)Configuration L. diaphragm No NA 1-2 No No 2   5 NL R. diaphragm Yes NA3-4 No No   2-7 5-20 NL R. deltoid Yes  180 1 Yes PSW, fibs 0.7 5polyphasic R. biceps No NA NA Yes PSW, fibs NA NA NA R. extensor carpiYes <200 2 Yes PSW, fibs, 1.5-2 5 polyphasic radialis CRD R. vastusmedialis Yes 200-500 1-2 Yes PSW, fibs 1.5-2 4 NL

EMG characteristics of key muscles in response to commands tovoluntarily move the muscle group. Columns indicate muscle groupsrecorded, presence of identifiable EMG response to activation request(voluntary response), delay in EMG activation compared to voluntaryactivation request (Response latency), number of motor units recruitedwith increasing effort to move (# Motor units recruited), presence ofactivity with insertion of the EMG needle (Insertional activity),presence of spontaneous resting activity (Spontaneous activity), andcharacteristics of motor units (Motor unit characteristics: Amplitude,Duration, and Configuration). Abbreviations: positive sharp waves (PSW);fibrillations (fibs); complex repetitive discharge (CRD); normal (NL);not assessed (NA); millivolts (mV); millisecond (ms).

5. Quality of Life Assessment. Table 5 lists the responses the patientmade in 2002 to questions about quality of life. Overall, recoveryimpacted many domains of daily living, but the changes perceived aslife-altering were: avoidance of sickness and sick days; improvedability to anticipate stable health and the ability to fulfill work andfamily obligations; improved health; improved weight control; moreattainable life goals; improved ability to breathe without a ventilator;positive impact on the family; greatly improved economics because ofreduced medical costs, fewer lost work days; enjoyment of beinghealthier; greater enjoyment of leisure activities; improvedproductivity; knowledge that progress had replaced regression; lessspasticity; and greater hope for additional recovery. Thesemi-quantitative quality-of-life measures that are available because oftheir limited ability to detect the impact of such a recovery.

Example 2 CNS Regeneration in Rats

Subjects, animal care and surgery: Thirty adult Long Evans female rats(275±25 g; Simonsen, Gilroy, Calif.) were housed (12:12 h light:darkcycle) and treated in accordance with the Laboratory Animal Welfare Act,and Guidelines/Policies for Rodent Survival Surgery (Animal StudiesCommittee of Washington University in St. Louis).

Six groups were examined using two survival intervals following the BrdUpulse label, either immediate or 7 days later. All 30 rats received FESimplants. Twenty-four rats received a spinal cord injury 3 weeks priorto FES device implantation. Six additional rats were uninjured controls.In half the animals (n=15) the FES systems were activated. FIG. 7( a)shows a schematic drawing of the experimental rat model showing therelationship between the injury and stimulation level.

FIG. 7( b) shows the 43-day time line for the experiment in which twelveLong Evans adult female rats received a complete transection of thespinal cord by suction ablation of two levels (T8-9). To simulate achronic type of injury we waited three weeks before the FES devices wereimplanted. Following a three-day post-surgical interval the devices inhalf of the rats were activated for 3 hours per day for 12 days. Betweenday 7 and day 12 of the FES schedule, we conducted a daily BrdU pulselabeling protocol. Two hours after the last BrdU injection all rats weresacrificed.

Bladders were expressed 3 times daily until recovery of reflex emptying.To prevent autophagia, rats were fitted with 10.5 cm plastic collars(Ejay, Glendora, Calif.), and housed individually with absorbent bedding(ALPHA-Dri™, Shepherd, Kalamazoo, Mich.) and nesting material.Individuals blinded to the treatment, and independent of the surgicalteam handled each rat for 5 min each day.

Spinal cord injury: Rats were anesthetized (75 mg/kg Ketaset®, 0.5 mg/kgDomitor®, i.p.), and a laminectomy was done at T7-T9. Through a 1 mmdura incision, 1 mm of the spinal cord was removed under microscopiccontrol via suction utilizing a BARON® suction tube (Roboz, Rockville,Md.). The dural opening was covered with fascia and the muscle andoverlying skin closed with layered sutures. Anesthesia was reversed byAntisedan® 1 mg/kg. This method of suction ablation minimized bleedingcompared to surgical blade transection, maintained the integrity of thedura and major blood vessels, and resulted in clean cord wound borders.

Stimulator and electrode implantation: Three weeks following SCI therats were re-anesthetized, a midline incision was made over the lowerback and a 2-channel battery powered electrical stimulator (Jarvis,University of Liverpool) and current return electrode were implanted ina subcutaneous pocket. A 0.5 cm incision was made on the lateral aspectof both shanks and stainless steel wire electrodes were tunneledbilaterally underneath the skin from the stimulator site, and suturedinto the tibialis anterior muscle adjacent to the common peroneal nerve.Intra-operative test stimulation ensured proper peroneal nerveactivation.

Electrical stimulation paradigm: FES devices were activated daily for 19days between 24 and 43 days after injury (starting at 3 days followingimplantation), for three 1 h sessions during each 9 h workday (FIG.11B). An additional group of 6 rats received the identical FES implantand activation pattern but were not injured. The pattern of stimulationwas 1 s stimulation of one common peroneal nerve, followed by 1 s ofrest, then the other common peroneal nerve was stimulated for 1 s,followed by 1 s of rest and the cycle repeated. Stimuli were 3 V 200 μsmonophasic pulses delivered at 20 Hz between the electrode over thenerve and the return electrode in the lower back and were expected toactivate large myelinated fibers within the common peroneal nerve, butnot small unmyelinated nociceptive fibers. The nerve stimulationproduced alternating flexion of the hindlimbs that crudely approximatedbilateral stepping-like hindlimb movements.

Bromodeoxyuridine injection paradigm: Newborn cells were identified bydaily injections, beginning day 31-36 after injury, with BrdU (50 mg/kgi.p.), a brominated DNA building block that is selectively incorporatedinto replicating DNA during the S phase of the cell cycle.

Tissue processing: Two hours after the final BrdU injection, half of therats (n=15) were deeply anesthetized and perfused intracardially with0.1 M PBS for 5 min followed by 4% paraformaldehyde (Sigma) for 15 min.One week after the final BrdU injection the remaining rats weresacrificed (n=15).

Immunohistochemistry: Every 6th section (40 μm) from spinal cord levelsC2, T1, T7, T11, L1, and L5 was selected for anti-BrdUimmunohistochemistry. Sections were incubated in 2 N HCl for 60 min at37° C., transferred to 0.1 M Borate buffer (pH 8.5) for 20 min, andrinsed with PBS. Non-specific labeling was blocked with 0.1% BSA in 0.1%Triton X-100/PBS for 60 min. A mouse monoclonal anti-BrdU antibody(1:600; Roche, Mannheim, Germany) was incubated with the tissueovernight at 4° C. Then tissue was treated with a CY3-conjugatedsecondary antibody (1:2000; Jackson, West Grove, Pa.) in 2% normal goatserum (NGS) for 60 min.

For co-labeling of the BrdU positive cells, sections were fixed with 4%paraformaldhyde for 30 min after the CY3-conjugated antibody wasapplied. They were then permeabilized when appropriate with 0.1%Triton-X-100 for 60 min, and blocked with 2% NGS for 60 min. Primaryantibodies diluted in 2% NGS were applied to the sections for 2 hours.Antibodies used included: rabbit anti-NG2 (1:250, Chemicon, Temecula,Calif.), mouse anti-Nestin (1:8, Developmental Studies HybridomaBank—DSHB), rabbit anti-GFAP (1:4, Diasorin, Stillwater, Minn.), mouseanti-APC-CC1 (1:20, Oncogene, Cambridge, Mass.), mouse anti-ED1 (1:100,Serotec, Raleigh, N.C.), mouse anti-OX42 (1:100, Serotec), sheepanti-Glut-1 (1:30, Biodesign, Saco, Minn.), rat anti-CD31 (1:20, BDPharmingen, Lexington, Ky.), mouse anti-NeuN (1:200, Chemicon), mouseanti-TUJ1 (1:200, Babco, Richmond, Calif.), guinea pig anti-doublecortin(1:3000, Chemicon), and mouse anti-PSA-NCAM (1:8, DSHB). Secondaryantibodies (1:300, Molecular Probes, Eugene, Oreg.) were conjugated toAlexa 488. Primary and secondary control slides were included with eachstain series.

Quantification of BrdU positive cells: The number of BrdU-labeled cellsas a function of spinal cord level was measured in a blinded fashion.Images were acquired on an Olympus IX70 Microscope equipped with aMagnafire® camera and quantitative counts were performed using acomputer assisted software package (Stereoinvestigator™,Microbrightfield Inc., VT). For unbiased stereological counts ofproliferating cells the indicator fractionator method was used. Theestimate of BrdU positive nuclei was based on counts of nuclei in aknown fraction of the volume of the region under consideration. Alabeling index was calculated by taking the total number of BrdUpositive nuclei per volume and dividing it by the total number ofHoechst 33342 (Molecular Probes) labeled nuclei per volume acquired fromcorresponding spinal cord sections. Sections were viewed under 200×, andsystematically scanned by moving in a raster pattern with the aid of amechanical stage (Ludl, Hawthorne, N.Y.). Stereoinvestigator™ softwaresuperimposed a sampling grid with an unbiased counting frame (x=122 m,y=110.1 m) on the image. The sampling frame was focused through thesection at a known distance and BrdU positive nuclei were marked insidethe counting frames. Based on these markers, area measurements, anddefined volumes, the software calculated the total number of BrdUpositive nuclei. To determine the phenotype, cells were evaluated forcolabeling with each phenotypic marker. For each BrdU positive cell, thecomplete cell nucleus was followed through the z-axis, and only cellswith a well-circumscribed immunopositive cell body were consideredpositive for a particular phenotype.

Statistical analysis: Comparison between experimental groups wasconducted by two-way ANOVA using SigmaStat® applying Tukey post-hoctests. Comparisons were made for the total number of BrdU positive cellsand phenotypic markers between groups and within groups by spinal cordlevel. For all statistical analyses significance was accepted at p<0.05with Bonferroni correction for multiple tests.

FES Increased the Number of BrdU Positive Cells Below the Level ofInjury

FES produced a substantial increase in the density of new cellbirth/survival at the lumbar levels of the spinal cord, consistent witha robust projection of the peroneal nerve to the lumbar spinal cord. Therostral-caudal selectivity of the effects of FES-induced hindlimblocomotion on cell birth/survival helps exclude possible globalmechanisms such as metabolism, blood borne and other systemic factors,and strengthens support for local mechanisms associated with increasedneural activity. Electrical fields in the range of 300 V/m can inducechanges in neural cell function. Based upon the 100× larger magnitude ofelectric field required to produce direct effects in other studies,compared to the very small electric field experience by the spinal cordin these studies (about 5 V/m), the neuronal activity rather than directelectric field effects is demonstrated to be responsible for theobserved effects.

The rat spinal cord was completely transected by suction ablation atT8/T9 to minimize intra-animal variability that might occur with lesscomplete lesions, but this model limited analysis of functionalrecovery. Previous studies indicate that thoracic separation of thespinal cord produces reduced neural activity below the lesion level andthat peripheral nerve stimulation or gait activity enhances activity atthe respective cord level. A chronic injury was chosen because it ismost relevant to human regeneration, the blood brain barrier is patent,and injury-induced cellular proliferation is normalizing.

A stimulation protocol of three one hour periods of phasic activity perday was based on previous findings that at least one hour of gaittraining per day was sufficient to double the number of progenitorderived neurons in the hippocampus of normal mice. Alternatingstimulation of the common peroneal nerve stimulation producedalternating flexion of the hindlimbs that crudely approximated bilateralstepping-like movements of the hindlimbs.

The newborn cells mainly expressed markers of tripotential and glialprogenitor cells, as well as astrocytes and oligodendrocytes. The numberof BrdU labeled microglia, macrophages or endothelial cells was smalland did not substantially contribute to the increased cellbirth/survival at L1 and L5, levels distant from the injury site (T8/9).This conclusion is consistent with the very low contribution (fewer than1.5%) of microglia and endothelial cells to total BrdU labeled cells inthe normal spinal cord.

For unbiased quantitative analysis, advanced stereological methods wereused that are well accepted in the field. The effect of FES was robustand selective and an effect of FES was not observed in normal uninjuredrats. The validity of the stereological measures is supported by thefact that the total number of cells (labeling index: 2.61% of totalnuclei) of newborn cells distant from the injury (e.g., C2) correlateclosely with values obtained with similar BrdU labeling methods andstereological analysis in the normal rat spinal cord observed in otherlabs.

FES Increased Tripotential Progenitor Birth after SCI

A five-day interval of BrdU labeling will primarily label proliferatingcells that do not have time to differentiate into mature cells,especially neurons. No evidence of new neuron birth was present in thespinal cord at day 0 or 7 after BrdU injection and this is consistentwith previous data demonstrating the absence of new neuron birth in theadult spinal cord.

FIG. 8 shows new cell birth in the adult spinal cord after chronic SCI.Panels A & B show 40 μm coronal sections of the spinal cord at the C2(A) and L1 (B) levels, which were immunolabeled with mouse anti-BrdUantibody. Sections were reconstructed using StereoInvestigator® fromindividual images at 20× magnification. Zones where the majority of BrdUpositive cells were found are indicated with arrows. Square letteredinserts in panels A & B correspond to magnified images in panels C-F.Although the greatest number of BrdU positive cells was found in thewhite matter (C), BrdU positive cells were also present in grey matter(D-E) and around the central canal (F). Scale bar in panels A-B is 1 mm,and scale bar in panels C-F is 50 μm.

FIGS. 9( a) and (b) show that FES promoted new cell birth/survival inthe damaged spinal cord. Quantitative counts of BrdU labeled cells wereperformed using stereological methods in coronal spinal cord sections(C2-L5). Two-way ANOVA demonstrated the effects of treatment and spinallevel (within groups, p<0.05). (A) FES induced a robust and selectiveincrease in new cell birth reserved to lower lumbar segments thatpredictably experienced increased activity from the patterned FESinduced stepping-like limb movements. (B) This effect persisted in thecell survival group 7 days later (*p<0.05; **p<0.001, FES vs. control).

FIG. 10, panels (a)-(l) shows BrdU colocalization with cell specificmarkers. Confocal microscopic images from an FES treated animal atlumbar level L5, two hours after the last BrdU injection. (A-C) Nestinimmunoreactivity (blue) was found predominantly in white matter andlabeled cells exhibited bipolar and multipolar morphology. Some of theNestin+ cells colabeled with GFAP (green, arrowhead), which mayrepresent reactive astrocytes, however, fewer than 2% double labeledwith BrdU (red). Thus, BrdU+/Nestin+ cells are proliferatingtripotential progenitors rather than reactive astrocytes. (D-F) SomeBrdU+/Nestin+ cells double labeled with NG2 (green, arrowhead). Thesecells were usually located near the pial layer and showed bipolarmorphologies. (G-I) Macrophages, here labeled with anti-ED1 (green),were located throughout the injured spinal cord. Fewer than 3% wereBrdU+ and were only found at levels close to the injury site. NoNestin+/ED1+ cells were identified. (J-L) Throughout the spinal cordparticularly in white matter (J=level C2, K=level L5) a small subset ofBrdU+ cells that co-labeled with the oligodendrocyte marker APC-CC1(green, arrowhead) was found. This ratio increased rapidly when observed7 days after the last BrdU injection suggesting that many glialprogenitors turn into oligodendrocytes. (L) Single confocal section of aBrdU+/APC-CC1+ cell. Scale bar (A-K)=50 μm, (L)=10 μm.

FIG. 11, panels (a)-(e) shows quantification of NG2 colocalization withBrdU following SCI at 2 hours after last BrdU injection. NG2immunoreactivity was used to classify BrdU+ cells as glial progenitorcells rostral (A) and caudal (B) to the lesion site. NG2+ cellsdisplayed bipolar and multipolar cell morphologies predominantly in thewhite matter (arrows indicate individual BrdU+/NG2+ cells). Panels C-Edepict single confocal sections of each marker for such an individualcell. The distribution pattern of Brdu+/NG2+ cells did not vary amongstgroups. However there was a significant decrease of BrdU+/NG2+immunoreactivity below the level of injury (47±3% at T1 and 51±3% at L5in Control; 33±3% at T1 and 39±3% at L5 in FES treated animals) implyingthat there were fewer glial progenitors newly generated. Two-way ANOVAdemonstrated the effect of spinal level (*p<0.05; **p<0.001, T1 vs. L5).Scale bar (A,B)=50 μm, (C-E)=10 μm.

FIG. 12, panels (a)-(e) shows quantification of GFAP colocalization withBrdU in the injured spinal cord at 2 hours after last BrdU injection.GFAP immunoreactivity was used to classify BrdU+ cells as astrocytesrostral (A) and caudal (B) to the lesion site. Labeled cells displayedpredominantly multipolar cell morphologies (arrows indicate individualBrdU+/GFAP+ cells). Panels C-E depict single confocal sections of eachmarker for an individual astrocyte. The distribution pattern of GFAP+cells did not vary amongst groups. An effect of spinal level on theBrdU+/GFAP+expression was not identified. Scale bar (A,B)=50 μm,(C-E)=10 μm.

FIG. 13, panels (a)-(e) shows quantification of Nestin colocalizationwith BrdU in the injured spinal cord at 2 hours after last BrdUinjection. Nestin immunoreactivity was used to classify BrdU+ cells astripotential progenitor cells found rostral (A) and caudal (B) to thelesion site (arrows indicate individual BrdU+/Nestin+ cells). Some ofthe Nestin+ cells show morphologic signs of reactive astrocytes(asterisk). However, fewer than 2% double labeled with BrdU. Panels C-Edepict single confocal sections of a typical Nestin+/BrdU+ cell. Thedistribution pattern of Nestin+ cells did not vary amongst groupsrostral to the lesion site. However, as seen with NG2 (FIG. 4) there wasan effect of spinal level (Two-way ANOVA, **p<0.001, T1 vs. L5). Mostimportantly, FES doubled the tripotential progenitor cell birth at L5.Scale bar (A,B)=50 μm, (C-E)=10 μm.

Quantitative analysis was conducted of progenitor phenotype at spinallevels T1 and L5 equidistant from the site of cord transection whichenabled comparison of cell phenotypes in a region where FES had noeffect and a region where the effects of FES were robust.

There were no quantitative differences in NG2+, GFAP+, or APC-CC1+ cellnumbers, however, the number of Nestin+ cells doubled from 10±3% to19±2% with FES treatment at level L5. Triple labeling procedures(BrdU/GFAP/Nestin) were used and confocal microscopy to rule out thepossibility that the increase in Nestin expression was due to reactiveastrocytosis caused by SCI or FES. However, the ability to identify thephenotypes of the majority of cells contributing to the enhanced cellbirth was limited as in previous studies.

Cell Survival was Decreased Below the Level of Injury

The effect of FES to increase the numbers of BrdU-labeled progenitorspersisted for at least one week following labeling. However, FES did notalter the percentage of surviving BrdU labeled cells. Seven days afterBrdU labeling, cell survival in both groups was significantly decreasedat all levels below the injury site (T11, L1, L5). In contrast, cellsurvival above the level of injury (C2, T7) was largely unchanged. AfterSCI, neural activity is dramatically reduced in regions below the injurylevel. The present invention demonstrates, however, that FES appears topreferentially affect cell birth rather than survival. It is likely thatactivity may be required for optimal differentiation and survival ofprogenitors, and the limited survival effect of FES treatment mayreflect a quantitative limit of activity induced by the present FESparadigm.

Implications of FES-Induced Neural Progenitor Proliferation onRegeneration and Recovery after SCI

The present invention represents the first demonstration that FES canenhance cellular generation in the injured adult CNS. Althoughelectrical stimulation is used clinically to attempt to enhance bonefusion and stimulate peripheral nerve growth, it has not been applied toCNS regeneration. The magnitude of the cell birth/survival response toFES in the present study was surprisingly large (61% to 77%). Based onthe volume of the entire adult rat spinal cord (estimated by volumedisplacement; 506±7 mm³, density=1.21 g/cm³) and the estimates of FESinduced cell birth, at level C2 approximately two new cells are born persecond in the current studies. Therefore, a million new cells were bornin just under one week, a number equivalent to numbers commonlytransplanted in the injured spinal cord for repair purposes. Harnessingthe potential of these endogenously born cells therefore represents arational approach to self-repair of the damaged CNS. Experiments foundin the Examples section of the present invention were designed tooptimize the experimental analysis of one index of regeneration and newcell birth.

The data suggests that FES may enhance recovery after chronic spinalcord injury. FES dramatically increases cell birth/survival in theinjured spinal cord may be one factor that could contribute tofunctional recovery. Activity-based recovery applications may be animportant long-term therapeutic target for individuals with SCI.

The effect of stepping-like limbs movements, produced by alternatingbilateral FES of the common peroneal nerve on cell genesis at six levels(C2, T1, T7, T11, L1, and L5) proximal and distal to complete spinaltransection, was examined. FIG. 11 illustrates the experimental outlineand the topographic relationship between the injury level and the FESstimulation level.

FES Reduced the Incidence of Medical Complications

The incidence of bladder infections was reduced in groups that receivedFES (incidence of 85±8% in control versus 65±10% in FES treated animalsover 12 days; p<0.05, Students t-test, n=12 per group). Bladderinfections were defined by onset of bloody or cloudy urine coincidingwith presence of red and white blood cells, struvite crystals, andepithelial cells in the urine sediment. Neither group showed any signsof autophagia, pain, or inflammation.

The Majority of Newly Born Cells were Located in White Matter

Anti-BrdU labeled cells indicative of new cell birth occurred in thespinal cords of both control and stimulated rats. Although it ispossible that BrdU may integrate into apoptotic cells as a result of DNAdamage repair, BrdU does not integrate in detectable levels into adultcells induced to undergo necrotic or apoptotic death. These data are inaccordance with observations in this study that BrdU positive cells donot colabel with anti activated Caspase-3 antibody, a marker of cellsdying by apoptosis (data not shown). Thus, the BrdU positive nucleievaluated in this study represent dividing cells.

The majority of labeled cells were found in the white matter rather thanthe gray matter, in keeping with previous reports of new cell birth inthe uninjured adult rats spinal cord. Anti-BrdU labeled cells werepresent in gray matter, particularly the dorsal horns, and a smallnumber of labeled cells were present around the central canal. The datamay reflect migration and do not directly assess the location of thecell birth.

FES Produced a Selective and Robust Enhancement of New CellBirth/Survival

Quantitative stereological analysis indicated that both spinal level andpresence of electrical stimulation were significant factors indetermining the numbers of BrdU labeled cells. A dramatic andstatistically significant increase in cell birth was observed in the FESgroup selective to the lumbar cord, the area expected to have enhancedactivity as a result of the FES-induced hindlimb movements (FIG. 8).Cell birth in the FES group increased by 61±18% at L1 and 77±11% at L5(labeling index, control vs. FES, n=6 per group: L1: 2.58 vs. 4.16,p=0.02, L5: 1.86 vs. 3.29, p<0.001). Comparable numbers of new cellsbetween groups were present at T11 (4.17 vs. 3.83 cells/mm³, p=0.48) andabove the transection (T1, T7 and C2). Importantly, the effect of FESpersisted when cell survival was examined 7 days after the last BrdUinjection (labeling index, control vs. FES, n=6 per group: L1: 1.62 vs.2.29, p<0.001, L5: 1.58 vs. 2.39, p<0.001).

FES Had No Effect in the Healthy Spinal Cord

To set the baseline of cell birth in Long Evans rats and to evaluate theinteractive effects of injury and FES we compared the results to a group(n=6) of non-injured rats that received the exact FES treatment and BrdUinjection paradigm. The animals were sacrificed 2 hours after the lastBrdU injection. There was no difference in number of BrdU positive cellsbetween groups at any level (labeling index, control vs. FES, n=3 pergroup: C2: 1.15 vs. 1.17, T1: 1.2 vs. 1.0, T7: 1.18 vs. 1.05, T11: 1.12vs. 1.32, L1: 1.17 vs. 0.9, L5: 0.83 vs. 0.8). There were significantlyfewer cells born in the normal cord than in the injured spinal cord(labeling index, SCI control vs. NO injury control: C2: 2.3 vs. 1.2; T1:3.0 vs. 1.2; T7: 4.1 vs. 1.2; T11: 4.2 vs. 1.1; L1: 2.6 vs. 1.2; L5: 1.9vs. 0.8).

Most BrdU Labeled Cells were Found Surrounding the Lesion Site

The injury associated pattern of cell birth/survival above the lesionwere similar between the two groups and were consistent with previouswork demonstrating that SCI induces new cell birth (FIG. 13). There wasa significant increase in cell birth/survival surrounding the spinalcord lesion compared to distant C2 and L5 levels (C2 vs. T7: 65±13%; C2vs. T11: 58±9%; L5 vs. T7: 113±9%; L5 vs. T11: 107±12%; Two way ANOVA:all comparisons shown above were significant p<0.05, n=6 in the controlgroup). In the FES treated group similar differences were observed incell numbers above the lesion but not below (FIG. 9).

BrdU Labeled Cells Expressed Mainly Glial and Progenitor Markers

Phenotypic analysis of anti-BrdU labeled cells in both groups indicatedthat the majority of cells born and evaluated immediately following the5 day BrdU pulse labeling were neural cells, especially in segmentsdistant to injury (C2, T1, L1, and L5). Anti-BrdU labeled cellsexpressed markers of tripotential progenitors (Nestin), glialprogenitors (NG2), astrocytes (GFAP) and oligodendrocytes (APC-CC1).There was no quantitative difference in NG2 (49±3% at T1; 36±3% at L5),GFAP (45±3% at T1; 40±3% at L5), or APC-CC1 (fewer than 5%) expressionbetween groups (FIG. 14-16). However, the number of Nestin positivecells nearly doubled with FES treatment from 10±3% to 19±2%, level L5.To distinguish between BrdU/Nestin positive tripotential progenitors andproliferating postmitotic astrocytes, triple labeling procedures wereperformed with GFAP. The majority (>90%) of BrdU/Nestin positive cellsdid not express GFAP. Therefore, most of the phenotypically identifiablecells are tripotential progenitors (FIG. 10). The limited ability toidentify the majority of increased numbers of cells is in keeping withsimilar low percentages in previous studies.

Microglia, Macrophages, and Endothelial Cells Minimally Contributed toIncreased Cell Birth/Survival Surrounding the Lesion Site

To examine the possibility that other non-neural cell types contributedto the total cell birth/survival measured in this study, the presence oftissue macrophages were determined, and microglia using the markeranti-ED1 and OX-42 (FIG. 10). ED-1 and OX-42 labeled macrophages andmicroglia surrounding the injury site and their numbers rapidly taperedas a function of distance from the injury site. Macrophages could alsobe easily distinguished by their characteristic cell body shape andeccentric nuclei and their bifringent inclusions. Double labeling withanti-BrdU revealed that a very small number of these cells were BrdUpositive (fewer than 2%). Anti-BrdU labeling was also occasionallyobserved in endothelial cells or pericytes of blood vessels (identifiedby characteristic morphology, location and immunoreactivity to specificmarkers (anti-CD31, anti-Glut-1)), but the total number of cells wassmall (fewer than 1.5%) and mostly restricted to the levels surroundingthe injury site (T8/T11). Thus, microglia, macrophages, and endothelialcells did not contribute substantially to the number of BrdU labeledcells measured at levels distant from the injury (C2, T1, L1, and L5).

Absence of Neurogenesis after Spinal Cord Injury

Despite a careful search for cells double-labeled with anti-BrdU andearly or late neuronal markers (anti-NeuN, anti-TUJ1, anti-PSA-NCAM, andanti-doublecortin) newborn neuronal phenotypes were not identified. Itis believed that the spinal cord was examined at too early a point inthe recovery to see direct evidence of neurons, and that later studiesexamining later stages in recovery will reveal the presence of neuronsconsistent with increased birth and survival of neural cells and withrecovery of function.

Example 3 Functional Reorganization and Stability of the CNS in theHuman Subject

The functional organization of somatosensory and motor cortex wasinvestigated in an individual with a high cervical spinal cord injury,the human subject of Example 1, supra. As described above, the subjectdemonstrated a 5-year absence of nearly all sensory and motor functionat and below the shoulders, and a rare and surprising recovery of somefunction in years 6-8 after intense and sustained rehabilitationtherapies. The results are described fully in Corbetta et al, Proc.Natl. Acad. Sci. 99: 17066-71 (Dec. 24, 2002), which is hereinincorporated by reference in its entirety, together with the primaryreferences contained therein.

Functional magnetic resonance imaging (fMRI) was used to study thesubject's brain activity in response to vibratory stimulation andvoluntary movements of body parts above and below the lesion. Noresponse to vibratory stimulation of the hand was observed in theprimary somatosensory cortex (SI) hand area, which was converselyrecruited during tongue movements that normally evoke responses only inthe more lateral face area. This result suggests SI reorganizationanalogous to previously reported neuroplasticity changes afterperipheral lesions in animals and humans. In striking contradistinction,vibratory stimulation of the foot evoked topographically appropriateresponses in SI and second somatosensory cortex (SII). Motor cortexresponses, tied to a visuomotor tracking task, displayed a near typicaltopography, although they were more widespread in premotor regions.These findings are consistent with preservation of motor and somesomatosensory cortical representations in the absence of overt movementsor conscious sensations for several years after spinal cord injury andhave implications for future rehabilitation and neural-repair therapies.

Severe sensory deprivation due to amputation or peripheral nerve damageprofoundly alters responsiveness and topographical organization ofprimary somatosensory cortex (SI). Less well known are the alterationsin cortical responses after spinal cord injury (SCI). Individuals withSCI, as opposed to those with amputations, retain a normal body, whichmay influence cortical reorganization significantly, especially inindividuals with partial SCI who have surviving fibers and potentiallysome functional connections across the level of damage. It will becomean important practical issue to assess cortical responsivenessimmediately after damage and in the course of recovery if ongoingefforts for restoring function by transplantation or other means such asFES-evoked patterned movement, are successful. Functional MRI (fMRI)with blood oxygenation level-dependent (BOLD) contrast provides anoninvasive method to assess neuronal activity by monitoringtask-related changes in the local tissue concentration ofdeoxyhemoglobin.

These fMRI results provide mapping of cortical somatosensory-motor areasin the human subject. Given the unique medical history of the subject inincluding late partial recovery of function after being clinicallyassessed as motor complete and nearly sensory complete, the fMRI studysought to determine the topographical normality of the subject'ssomatosensory and motor cortical responses above and below the level ofdamage, and to investigate possible cortical reorganization subsequentto a late recovery from SCI.

Subjects. Example 1 describes in detail the clinical history of the50-year-old right-handed male who sustained a displaced C2 type IIodontoid fracture from an equestrian accident in 1995 at age 42. Noother permanent injuries, particularly a head injury, complicated theSCI. By clinical assessment, motor or somatosensory functions wereabsent below the lesion level for 5 years except for spotty sensation inthe left hemitorso. He is ventilator dependent with hypophonicvocalization due to impaired function of the chest diaphragm and musclesof vocalization. The control subject was a 23-year-old male who has anormal neurological and psychiatric history.

Visuomotor Tracking. Subjects were required to synchronize movements toa video image of a yellow-green tennis ball against a black background.Subjects viewed the image on a back projection screen, which was seen ina mirror mounted on the head coil. The ball (≈4° in diameter) jumpedregularly left/right of a fixation point (≈4° jumps, 0.83-Hz rate) toguide tongue left/right movements (tongue extruded and moved againstlips), jumped above/below the fixation point to guide left index-fingermovement (at metacarpal-phalangeal joint) and remained stationary forrest periods. Visual monitoring indicated that both subjectsconsistently followed the ball motion. Movement range and vigor wereless in the SCI subject. The left index finger was tested because theSCI subject sustained better following with this finger movement.Subjects could not see their finger during fMRI. For testing consistencyall tactile stimulation was also applied to the left extremities.

Vibrotactile Stimulation. A massage vibrator delivered suprathresholdtactile stimulation. The device, previously used in positron-emissiontomography studies, was made magnetic resonance-compatible by replacingthe electric motor with a pneumatic drive that was connected to a remoteair compressor. The vibrator delivered approximately 2-mm displacementvibrations centered on a base frequency of approximately 100 Hz. Thevibrator head was manually held against the left fingers and palm orsole of the left foot throughout stimulation and rest periods. Preciseskin displacements were unknown, although stimulus magnitudes probablyactivated most skin mechanoreceptors, adjoining deeper tissues, andproprioceptors throughout distal parts of the stimulated limb.

MRI Acquisitions. During MRI the SCI subject was ventilated, andphysiologic parameters were monitored continuously (Magnitude/Milleniumanesthesia monitoring, In Vivo Research, Orlando, Fla.) in a custommagnetic resonance-compatible setup (Shielding Resources Group, Tulsa,Okla.). All MRI used a 1.5-Tesla Magnetom Vision scanner and circularlypolarized head coil (Siemens, Erlangen, Germany). Structural 3DT1-weighted magnetization-prepared_rapid gradient echo MRI was acquired.fMRI used a custom T2* -weighted asymmetric spin-echo echo-planarsequence sensitive to BOLD contrast (repetition time=2,360 ms, T2*evolution time=50 ms, α=90°). During each fMRI run, 128 sets of 20contiguous, 6-mm thick slices were acquired parallel to the anteriorcommissure posterior commissure plane (3.75×3.75 mm in-plane voxelsize), allowing complete brain coverage. This protocol yielded images ofthe SCI subject without significant signal loss or distortions in thebrain despite surgical metal in the neck. Sensory and motor fMRI wereacquired in different imaging sessions by using 8-10 fMRI runs persession and 128 frames (346.75 s) per run. For sensory fMRI, four runswere obtained for each stimulated limb, and each run contained eightbaseline frames followed by 15 trials with three frames of stimulationalternating with five frames of no stimulation. For motor fMRI, four tofive runs were obtained for each task (tongue and finger), each runhaving 12 trials of five task frames alternating with five rest frames.

MRI Data Analysis. The cord was segmented from structural MRI for areameasurement and 3D-rendered display by using ANALYZE AVW 4.0 (MayoFoundation, Rochester, Minn.) and a Sun Fire V880 computer (SunMicrosystems, Santa Clara, Calif.). fMRI data were analyzed as describedin Burton et al., J. Neurophysiol. 87: 589-607 (2002), and Corbetta etal., Neuron 21: 761-73 (1998). General linear models estimated the BOLDresponses in each subject and each task (e.g., tongue movement) withoutassuming a hemodynamic response shape. BOLD time courses were estimatedin each voxel, over eight frames (21.67 s) for somatosensory tasks and10 frames (27.09 s) for motor tasks. fMRI data were smoothed with a 3D2-voxel Gaussian kernel and transformed to the Talairach atlas beforestatistical analysis. Statistical maps were based on cross-correlationbetween estimated BOLD time course and a reference hemodynamic responsefunction that was obtained by convolving a delayed gamma function with arectangular function representing task and control periods. The derivedt statistics per voxel were converted to normally distributed z scoresand corrected for multiple comparisons across the entire brain by usingdistributions obtained from Monte Carlo simulations (based on methodsdescribed in S. D. Forman et al., Magn. Reson. Med. 33:636-47 (1995).These images were inspected by using a threshold of P=0.05 for a z scorevalue of 4.5 over at least three face-contiguous voxels. The statisticalmaps were projected onto a standard brain atlas in both 3D view of thelateral hemispheric surface and 2D view of flattened cortex (Van Essenet al., Proc. Natl. Acad. Sci. USA 95: 788-95 (1998); seehttp://stp.wustl.edu/resources/caretnew.html).

Structural MRI of the Cord. T1-weighted MRI in the SCI subject showedgreater than 75% loss in cross-sectional tissue area throughout the C2region.

FIG. 14 shows a T1-weighted MRI of cervical SCI in SCI subject. The toppanel shows longitudinal 3D-rendered image (view from behind) oflower-brainstem and cervical spinal cord segments from the tip of the C2odontoid process through the bottom of the C2 vertebral body. The middlepanel shows selected low-magnification images through the zone of injuryshow the small size of the cord relative to the spinal canal (SC).Images are transverse to the cord's long axis and at levels 40, 46, and51 mm below the cerebellar tonsils (Left, Center, and Right,respectively). The bottom panel shows higher-magnification view of thesame three transverse images show focal regions of low T1-weightedsignal that are consistent with chronic tissue damage (myelomalacia) orscarring. The location and shape of these sites vary and include acentral oval (red arrow), a cleft (blue arrow), and several peripherallesions (yellow arrows). L indicates left, R, indicates right.

Additionally, there were multiple focal areas of tissue damage(myelomalacia) in different parts of the remaining spinal cord. Thecontinuity of white-matter pathways across the lesion cannot bedetermined from these images alone, particularly given the small cordsize and the multiple areas of damage within and across levels.

Clinical History of SCI Patient. Initial recovery of motor andsomatosensory function began in year 2000 after several years ofstandard physical therapy including passive range of motion andsupported standing. The recovery accelerated through 2002 after thesubject was enrolled in a more intense regime of physical therapy.Physical therapy included thrice-weekly, hour-long sessions offunctional electrical stimulation of leg muscles that was computersynchronized to drive an exercise bicycle as sown in FIG. 2. Frequentstandard clinical examinations in the last 2 years documentedself-initiated small movements with the left index finger, right wrist,and more recently, lower extremities. In parallel, the SCI subjectreported feeling strong sensation upon tactile stimulation and passivemovement of the upper and lower extremities. Sensations from the lowerextremities were stronger. He had good accuracy in roughly localizing atactile stimulus on the hand or foot including dorsal and volarsurfaces. He also could identify a stimulated finger or toe (fingersbetter than toes).

Motor Tracking Tasks. FIG. 15 shows BOLD responses to the visuallyguided motor tasks on 3D and flattened representations of a standardbrain. 3D and 2D flattened views of an atlas brain are shown withprojected BOLD responses for visuomotor tracking task; the color scaleindicates z scores. FIG. 15A shows a control subject, while FIG. 15Bshows the subject with SCI.

Tongue movements in the control subject activated a ventral segment ofprecentral gyrus, central sulcus, and postcentral gyrus bilaterally(FIG. 15A, SMfc). These regions likely correspond to the primary sensoryand motor cortex face area. Recruitment of SI in a visuomotor trackingtask probably reflects tactile and proprioceptive sensory feedbacksignals associated with normal movements. Significant activity alsooccurred in medial frontal cortex along the cingulate sulcus(supplementary motor area/anterior cingulate) and in visual occipitalcortex.

Left index-finger movements produced the strongest activation in amiddle segment of the contralateral (right) precentral gyrus_sulcus(FIG. 15A, Mh). This response was approximately 2 cm superior to thefoci active during tongue movements. Smaller responses occurred in theright parietal operculum and bilaterally within the central sulcus andpostcentral gyrus (SI, area 3b); these likely related to sensoryfeedback.

Finally, bilateral responses were observed in supplementary motorarea/anterior cingulate similar to those observed for tongue movements.In the subject with SCI, BOLD responses during both motor tasks werestronger and more widespread than those observed in the control subject(FIG. 15 B vs. A). Tongue movements activated the face area of SI/M1 inventral precentral gyrus, central sulcus, and postcentral gyrus (primarysomatosensory-motor cortex, SMfc). Activity spread dorsally into thehand area and more extensively into adjacent regions such as the secondsomatosensory cortex (SII) and frontal operculum.

There was also strong activation of dorsolateral prefrontal cortex andsupplementary motor area/anterior cingulate. Left index-finger movementsintensely activated the contralateral M1 hand area (Mh), frontaloperculum, SII, cingulate cortex, and lateral and medial parietal cortex(FIG. 15B). There was no spread into the SI/M1 face area. Recruitment ofvisual cortex, both ventrally and dorsally, was much stronger in the SCIthan control subject and during finger than tongue movements in the SCIsubject (FIG. 15B). In one experiment, fMRI was acquired while the SCIsubject performed left finger movements without visual feedback for 20-sintervals. Voluntary movements were internally planned and executedafter a brief verbal “GO” signal. Performance was less accurate and lesssustained, and the subject reported more effort, compared withvisuomotor tracking. BOLD responses (movement vs. rest) were weaker andless localized in M1, and there was no recruitment of premotor orhigher-order regions (not shown).

Vibrotactile fMRI. FIG. 16 shows BOLD responses to vibratory stimulationof the left hand or left foot. 3D and 2D flattened views of the atlasbrain are shown, on which BOLD responses for sensory vibrotactilestimuli have been projected; the color scale indicates z scores. FIG.16A shows results for a control subject, and FIG. 16B shows those forthe Subject with SCI.

In the control subject (FIG. 16A), left hand and fingers vibrationactivated the contralateral (right) SI/M1 cortex. Responses occupied thedorsal segment of the postcentral gyrus extending into the centralsulcus and the precentral gyrus, thus identifying the normal location ofthe hand area (FIG. 16A, SMh). There was also bilateral activation ofSII. Left foot vibration evoked activity in the right medial frontalgyrus approximately 1.2 cm dorsal to the hand area (FIG. 16A Right).This response likely involved the SI/M1 foot area (SMft). SII wasactivated bilaterally. The subject with SCI reported a better sensationof vibration in the left foot than in the left hand. Left hand vibrationfailed to activate expected hand areas of SI/M1 in the right postcentralgyrus, central sulcus, and precentral gyrus but produced bilateralresponses in SII (FIG. 16B, Left).

Several other regions not recruited in the control subject responded inthe SCI subject. These regions included, in order of strength, thecontralateral (right) postcentral sulcus, and bilaterally, the posteriorpostcentral gyrus, and supramarginal gyrus (PoCs/5 mg); the ipsilateral(left) postcentral gyrus; and contralateral medial frontal gyrus nearthe SI/M1 foot area. The contralateral regions correspond to likelyhigher-order somatosensory areas. Left foot stimulation in the SCIsubject (FIG. 16B Right) elicited a normal contralateral response in theright medial frontal gyrus, central sulcus, and postcentral gyrus thatcorresponds to the normal SI/M1 foot area (SMft, compare FIG. 16 A withB). An ipsilateral SI response also occurred in the left hemisphere. SIIresponses again were located along the parietal operculum but wereweaker than during hand stimulation.

There was some recruitment of higher-order parietal regions in rightsupramarginal gyrus during foot stimulation. The fMRI from the SCI wasoverlaid with those of control subjects to compare somatosensory-motorcortex areas. FIG. 17A contrasts activation maps for left hand vibrationand tracking tongue movements in the control subject. FIG. 17B shows theabsence of a normal hand area in the SCI subject. FIG. 17C shows thatthe face area is similarly active in both subjects. However, the activeregion expands dorsally in the SCI subject where it encroaches onto thenormal hand area (FIG. 17D).

Functional MRI in the SCI subject with late partial recovery ofsensorimotor function revealed BOLD responses in the SI foot area duringvibratory foot stimulation, responses in higher-order somatosensoryareas during stimulation of the hand, and responses in motor cortexareas during finger movements. In contrast, no response was observed inthe S1 hand area upon vibratory hand stimulation. This region insteadwas recruited during sensory-motor stimulation of the tongue and lips.The brain responses to vibratory stimuli occurred despite greater than75% loss in spinal cord area and multiple regions of chronic damage inthe remaining cord. These responses indicate the existence of functionalneural connections that traverse the site of cord injury. The subject'slate clinical recovery is surprising given the 5-year history of novoluntary movements and nearly absent sensations from the extremities.Some brain responses were consistent with known neuroplasticity effectsdemonstrated mostly in animal studies, while others were entirelyunexpected and are consistent with preservation of normal topography andrecruitment of compensatory areas.

The S1 hand area responded to sensory stimulation of the face but notthe hand, which possibly reflects neural plasticity based on competitiveinteraction from a normally innervated face. This response of the(deafferented) hand area to (invading) face inputs might correlate withan enhanced resolution of tactile sensation in the face, as demonstratedon a much smaller spatial scale within the digit representation ofmonkey area 3b.

The reorganization possibly involved subcortical transneuronal changes,which are progressive and delayed. Cortical activity is likely toreflect this mechanism by initial silence followed by late recovery ofresponses. Similar to prior results in monkeys with long-term dorsalrhizotomies, expansion in the SCI subject involved SI hand arearesponses to stimulation in the lower face, which borders the thalamichand area. A conjunction of mechanisms is likely to be responsible forthe SI foot-area responses and the reported better sensations from thefoot. Absent was competition from intact representations because allcortical and subcortical areas adjacent to the foot were severelydeprived. This likely left the SI foot area accessible to any input thatwas conveyed through surviving fibers at the level of the injury (FIG.14).

Clinical changes accelerated after more intense rehabilitation involvingFES-evoked patterned movement of the lower extremities in accordancewith the methods of the present invention. The SCI subject was able tolocalize tactile stimulation on the hand, despite little evidence ofresponses in the SI hand area to intense tactile vibrations. Vibratorystimulation of the hand recruited SII and additional postcentral andposterior parietal somatosensory regions that normally do not respondduring passive stimulation but become active during attention-requiringtactile discrimination tasks. In addition, recruitment of more posteriormultimodal parietal regions was observed.

In contrast to the changes found in somatosensory maps, the primarymotor areas were more nearly normal despite years without movements.Finger movement activated a confined middle segment of M1, whereastongue movements, although activating hand SI areas, did not invadeadjacent M1 regions.

The secondary motor areas (premotor and cingulate cortex) were activatedmore in the SCI vs. control subject. Additionally, many higher-orderregions (e.g., posterior parietal, temporal, and dorsolateral prefrontalcortex) were recruited in the SCI subject, more for finger than tonguemovements. Although tongue movements use neurons above the lesion, it isnot surprising that an abnormally widespread network of activity wasevoked, because these movements require coordination of several musclegroups (e.g., diaphragm or accessory respiratory muscles) that haveC2-C5 myotomes at or below the lesion.

Coupling of visual and motor information aided motor performance and ledto stronger, more widespread activity compared with self-timedmovements. The differences in visually guided vs. nonguided movementscannot be explained solely by a greater effort or lack of proprioceptivefeedback, because these effects were similar in the two tasks. Thus, itis likely that the regular oscillation of the tennis ball image providednecessary timing and directional information to plan and execute moresustained and regular movements.

In studies of SCI without recovery, normal motor cortical responses havebeen found during attempted/imagined toe movements in 9/9 subjects withthoraco-lumbar paraplegia but weaker sensory cortical responses topassive unconscious mobilization in 3/9 subjects. Normal SII activationhas been described in 3/3 paraplegics but only weak SI foot activationin one subject. The data herein extend those studies by assessingpreservation and rearrangement of cortical topography (face vs. hand vs.foot) in the rare case of partial recovery years after SCI.

The finding of any normal topography in somatosensory and motor areas ofa tetraplegic is surprising given a long history of no sensations ormovements from below the injury. These fMRI findings were coincidentwith the extraordinary and intensive rehabilitation therapies includinga program of FES-evoked patterned movement in a patient with severe butnot total SCI.

The mere presence of these cortical responses years afterdeafferentation is consistent with the goal of therapeutic strategiesaimed at restoring spinal cord connections because they suggest thatrestoration of neural links across the lesion appear capable ofreestablishing motor and sensory functions.

Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present invention. However, theinvention described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description which do not depart from thespirit or scope of the present inventive discovery. Such modificationsare also intended to fall within the scope of the appended claims.

REFERENCES CITED

All publications, patents, patent applications and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentinvention.

1. A method for treating central nervous system (CNS) damage in amammalian subject in need of such treatment comprising: inducing atherapeutically effective amount of functional electrical stimulation(FES)-evoked patterned movement in a mammalian subject for a period oftime sufficient to regenerate neural cells; and monitoring the mammaliansubject for CNS neural regeneration or CNS motor function; wherein thepatterned movement occurs during exposure to FES; and at least a portionof lost motor function or sensory function of the mammalian subject lostis restored; and the CNS damage comprises (i) an acute trauma or (ii) achronic disease.
 2. The method of claim 1 wherein the acute trauma isselected from the group consisting of complete severance of the spinalcord, partial severance of the spinal cord, and stroke.
 3. The method ofclaim 1 wherein the chronic disease selected from the group consistingof multiple sclerosis, cancer, tumor metastasis, Huntington's Disease,Alzheimer's disease, amyotrophic lateral sclerosis, andneurodegenerative effects of aging.
 4. The method of claim 1 wherein thetherapeutically effective amount of FES-evoked patterned movementpromotes neural cell birth.
 5. The method of claim 1 wherein thetherapeutically effective amount of FES-evoked patterned movementenhances neural cell survival.
 6. The method of claim 1 wherein thetherapeutically effective amount of FES-evoked patterned movementenhances neural signal transmission capacity.
 7. The method of claim 6wherein the therapeutically effective amount of FES-evoked patternedmovement initiates synaptic changes in neural cells such that the neuralcells reconfigure with neural cells to form new synaptic connections inor around a site of CNS damage.
 8. The method of claim 1 wherein themammalian subject is motor complete or sensory complete.
 9. The methodof claim 1 wherein the neural cells comprise at least one oftripotential progenitor cells, glial progenitor cells, astrocytes andoligodendrocytes or any combination thereof.
 10. The method of claim 1wherein the FES-evoked patterned movement activates a central patterngenerator in the spinal cord.
 11. The method of claim 1 wherein thepatterned movement comprises at least one of walking, breathing, biking,punching, kicking, swimming, fist clinching, toe pointing, knee bending,hip flexing, sitting, standing, and jumping.
 12. The method of claim 11,wherein the FES is applied to at least one muscle or muscle groupselected from the group consisting of gluteals, paraspinal, abdominals,wrist extensors, wrist flexors, deltoids, biceps, triceps, hamstrings,and quadriceps.
 13. The method of claim 1, wherein the application offunctional electrical stimulation is applied to a muscle or muscle groupexternally.
 14. The method of claim 1, wherein the application offunctional electrical stimulation is applied to a muscle or muscle groupinternally.
 15. The method of claim 1, wherein the FES-evoked patternedmovement occurs for at least about one hour per day and at least threetimes per week.
 16. The method of claim 1 wherein said therapeuticallyeffective amount of FES-evoked patterned movement comprises deliveringelectronic stimulation at 3 V and 200 μs monophasic pulses delivered at20 Hz.
 17. The method of claim 1, wherein the neural cells are capableof transmitting an at least partially restored neural signal.
 18. Themethod of claim 17, wherein the at least partially restored neuralsignal begins in the brain of the subject and ends in a muscle incommunication with a peripheral nervous system nerve cell.
 19. Themethod of claim 18, wherein the at least partially restored neuralsignal ending in a peripheral nervous system nerve cell causes themuscle to contract.
 20. The method of claim 19, wherein inducing atherapeutically effective amount of FES-evoked patterned movementcomprises stimulation of peripheral nervous system sufficient to evoke acycle of neural signal activity in the peripheral nerves and in thespinal cord, followed by neural signal inactivity therein, and whereinthe cycle is repeated.
 21. The method of claim 20, wherein the neuralsignal cycle is sufficient to evoke the muscles to perform patternedmovement.
 22. The method of claim 21, wherein said patterned movementcomprises reciprocal movements.
 23. The method of claim 1, comprisingmonitoring the mammalian subject for CNS neural regeneration.
 24. Themethod of claim 23, wherein monitoring the mammalian subject for CNSneural regeneration comprises fMRI imaging.
 25. The method of claim 1,comprising monitoring the mammalian subject for CNS motor function. 26.The method of claim 1, wherein the patterned movement comprises bikingand the FES is applied to at least a gluteal muscle, a quadricep muscle,and a hamstring muscle.
 27. The method of claim 26 wherein theFES-evoked patterned movement occurs for at least about one hour per dayand at least three times per week.